Compositions and methods for inhibiting expression of the PCSK9 gene

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the proprotein convertase subtilisin kexin 9 (PCSK9) gene, comprising an antisense strand having a nucleotide sequence which is less than 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of the PCSK9 gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; and methods for treating diseases caused by PCSK9 gene expression by using the pharmaceutical composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/799,458, filed May 11, 2006; U.S. Provisional Application No.60/817,203, filed Jun. 27, 2006; U.S. Provisional Application No.60/840,089, filed Aug. 25, 2006; U.S. Provisional Application No.60/829,914, filed Oct. 18, 2006; and U.S. Provisional Application No.60/901,134, filed Feb. 13, 2007. The contents of all of theseprovisional applications are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), andits use in mediating RNA interference to inhibit the expression of thePCSK9 gene and the use of the dsRNA to treat pathological processeswhich can be mediated by down regulating PCSK9, such as hyperlipidemia.

BACKGROUND OF THE INVENTION

Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of thesubtilisin serine protease family. The other eight mammalian subtilisinproteases, PCSK1-PCSK8 (also called PC1/3, PC2, furin, PC4, PC5/6,PACE4, PC7, and S1P/SKI-1) are proprotein converges that process a widevariety of proteins in the secretory pathway and play roles in diversebiological processes (Bergeron, F. (2000) J. Mol. Endocrinol. 24, 1-22,Gensberg, K., (1998) Semin. Cell Dev. Biol. 9, 11-17, Seidah, N. G.(1999) Brain Res. 848, 45-62, Taylor, N. A., (2003) FASEB J. 17,1215-1227, and Zhou, A., (1999) J. Biol. Chem. 274, 20745-20748). PCSK9has been proposed to play a role in cholesterol metabolism. PCSK9 mRNAexpression is down-regulated by dietary cholesterol feeding in mice(Maxwell, K, N., (2003) J. Lipid Res. 44, 2109-2119), up-regulated bystatins in HepG2 cells (Dubuc, G., (2004) Arterioscler. Thromb. Vase.Biol. 24, 1454-1459), and up-regulated in sterol regulatory elementbinding protein (SREBF) transgenic mice (Horton, J, D., (2003) Proc.Natl. Acad. Sci. USA 100, 12027-12032), similar to the cholesterolbiosynthetic enzymes and the low-density lipoprotein receptor (LDLR).Furthermore, PCSK9 missense mutations have been found to be associatedwith a form of autosomal dominant hypercholesterolemia (Hchola3)(Abifadel, M., et at (2003) Nat. Genet, 34, 154-156, Timms, K. M.,(2004) Hum. Genet 114, 349-353, Leren, T. P. (2004) Clin. Genet. 65,419-422), PCSK9 may also play a role in determining LDL cholesterollevels in the general population, because single-nucleotidepolymorphisms (SNPs) have been associated with cholesterol levels in aJapanese population (Shioji, K., (2004) J. Hum. Genet. 49, 109-114).

Autosomal dominant hypercholesterolemias (ADHs) are monogenic diseasesin which patients exhibit elevated total and LDL cholesterol levels,tendon xanthomas, and premature atherosclerosis (Rader, D. J., (2003) J.Clin. Invest. 111, 1795-1803). The pathogenesis of ADHs and a recessiveform, autosomal recessive hypercholesterolemia (ARH) (Cohen, J. C.,(2003) Curr. Opin. Lipidol. 14, 121-127), is due to defects in LDLuptake by the liver, ARH may be caused by LDLR mutations, which preventLDL uptake, or by mutations in the protein on LDL, apolipoprotein B,which binds to the LDLR. ARH is caused by mutations in the ARM proteinthat are necessary for endocytosis of the LDLR-LDL complex via itsinteraction with clathrin. Therefore, if PCSK9 mutations are causativein Hchola3 families, it seems likely that PCSK9 plays a role inreceptor-mediated LDL uptake.

Overexpression studies point to a role for PCSK9 in controlling LDLRlevels and, hence, LDL uptake by the liver (Maxwell K. N. (2004) Proc.Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004) J.Biol. Chem. 279, 48865-18875, Park, S. W., (2004) J. Biol. Chem. 279,50630-50638). Adenoviral-mediated overexpression of mouse or human PCSK9for 3 or 4 days in mice results in elevated total and LDL cholesterollevels; this effect is not seen in LDLR knockout animals (Maxwell K. N.(2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al.(2004) J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol.Chem. 279, 50630-50638). In addition, PCSK9 overexpression results in asevere reduction in hepatic LDLR protein, without affecting IDLE mRNAlevels, SREBP protein levels, or SREBP protein nuclear to cytoplasmicratio. These results indicate that PCSK9, either directly or indirectly,reduces LDLR protein levels by a post transcriptional mechanism

Loss of function mutations in PCSK9 have been designed in mouse models(Rashid et al., (2005) PNAS, 102, 5374-5379, and identified in humanindividuals Cohen et al., (2005), Nature Genetics., 37.161-165. In bothcases loss of PCSK9 function lead to lowering of total and LDLccholesterol. In a retrospective outcome study over 15 years, loss of onecopy of PCSK9 was shown to shift LDLc lower and to lead to an increasedrisk-benefit protection from developing cardiovascular heart disease(Cohen et al. 2006 N. Engl. J. Med., 354, 1264-1272.). Clearly theevidence to date indicates that lowering of PCSK9 levels will lowerLDLc.

Recently, double-stranded RNA molecules (dsRNA) have been shown to blockgene expression in a highly conserved regulatory mechanism known as RNAinterference (RNAi), WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631. Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al). This natural mechanism hasnow become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

Despite significant advances in the field of RNAi and advances in thetreatment of pathological processes which can be mediated by downregulating PCSK9 gene expression, there remains a need for agents thatcan inhibit PCSK9 gene expression and that can treat diseases associatedwith PCSK9 gene expression such as hyperlipidemia.

SUMMARY OF THE INVENTION

The invention provides a solution to the problem of treating diseasesthat can be modulated by down regulating the proprotein convertasesubtilisin kexin 9 (PCSK9) by using double-stranded ribonucleic acid(dsRNA) to silence PCSK9 expression.

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the PCSK9gene in a cell or mammal using such dsRNA. The invention also providescompositions and methods for treating pathological conditions that, canmodulated by down regulating the expression of the PCSK9 gene, such ashyperlipidemia. The dsRNA of the invention comprises an RNA strand (theantisense strand) having a region which is less than 30 nucleotides inlength, generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an mRNA transcript of the PCSK9 gene.

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the PCSK9 gene.The dsRNA comprises at least two sequences that are complementary toeach other. The dsRNA comprises a sense strand comprising a firstsequence and an antisense strand comprising a second sequence. Theantisense strand comprises a nucleotide sequence which is substantiallycomplementary to at least part of an mRNA encoding PCSK9, and the regionof complementarity is less than 30 nucleotides in length, generally19-24 nucleotides in length. The dsRNA, upon contacting with a cellexpressing the PCSK9, inhibits the expression of the PCSK9 gene by atleast 40%.

For example, the dsRNA molecules of the invention can be comprised of afirst sequence of the dsRNA that, is selected from the group consistingof the sense sequences of Table 1 and Table 2 the second sequence isselected from the group consisting of the antisense sequences of Tables1 and Table 2. The dsRNA molecules of the invention can be comprised ofnaturally occurring nucleotides or can be comprised of at least onemodified nucleotide, such as a 2′-O-methyl modified nucleotide, anucleotide comprising a 5′-phosphorothioate group, and a terminalnucleotide linked to a cholesteryl derivative. Alternatively, themodified nucleotide may be chosen from the group of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. Generally, such modifiedsequence will be based on a first sequence of said dsRNA selected fromthe group consisting of the sense sequences of Tables 1 and Table 2 anda second sequence selected from the group consisting, of the antisensesequences of Tables 1, and Table 2.

In another embodiment, the invention provides a cell comprising one ofthe dsRNAs of the invention. The cell is generally a mammalian cell,such as a human cell.

in another embodiment, the invention provides a pharmaceuticalcomposition for inhibiting the expression of the PCSK9 gene in anorganism, generally a human subject, comprising one or more of the dsRNAof the invention and a pharmaceutically acceptable carrier or deliveryvehicle.

In another embodiment, the invention provides a method for inhibitingthe expression of the PCSK9 gene in a cell, comprising the followingsteps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA comprises at least two sequences that        are complementary to each, other. The dsRNA comprises a sense        strand comprising a first sequence and an antisense strand        comprising a second sequence. The antisense strand comprises a        region of complementarity which is substantially complementary        to at least a part of a mRNA encoding PCSK9, and wherein the        region of complementarity is less than 30 nucleotides in length,        generally 19-24 nucleotides in length, and wherein the dsRNA,        upon contact with a cell expressing the PCSK9, inhibits        expression of the PCSK9 gene by at least 40%; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of the        PCSK9 gene, thereby inhibiting expression of the PCSK9 gene in        the cell.

In another embodiment, the invention provides methods for treating,preventing or managing pathological processes which can be mediated bydown regulating PCSK9 gene expression, e.g. hyperlipidemia, comprisingadministering to a patient in need of such treatment, prevention ormanagement a therapeutically or prophylactically effective amount of oneor more of the dsRNAs of the invention.

In another embodiment, the invention provides vectors for inhibiting theexpression of the PCSK9 gene in a cell, comprising a regulatory sequenceoperably linked to a nucleotide sequence that encodes at least onestrand of one of the dsRNA of the invention.

In another embodiment, the invention provides a cell comprising a vectorfor inhibiting the expression of the PCSK9 gene in a cell. The vectorcomprises a regulatory sequence operably linked to a nucleotide sequencemat encodes at least one strand of one of the dsRNA of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of the ND-98 lipid.

FIG. 2 shows the results of the in vivo screen of 16 mouse specific(AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs directed against differentORF regions of PCSK9 mRNA (having the first nucleotide corresponding tothe ORF position indicated on the graph) in C57/BL6 mice (5animals/group). The ratio of PCSK9 mRNA to GAPDH mRNA in liver lysateswas averaged over each treatment group and compared to a control grouptreated with PBS or a control group treated with an unrelated siRNA(blood coagulation factor VII).

FIG. 3 shows the results of the in vim screen of 16 human/mouse/ratcrossreactive (AL-DP-9311 through AL-DP-9326) PCSK9 siRNAs directedagainst different ORF regions of PCSK9 mRNA (having the first nucleotidecorresponding to the ORF position indicated on the graph) in C57/BL6mice (5 animals/group). The ratio of PCSK9 mRNA, to GAPDH mRNA in liverlysates was averaged over each treatment group and compared to a controlgroup treated with PBS or a control group treated with an unrelatedsiRNA (blood coagulation factor VII).

Silencing of PCSK9 mRNA resulted in lowering total serum cholesterollevels.

The most efficacious in terms of knocking down PCSK9 message siRNAsshowed the most pronounced cholesterol lowering effect (around 20-30%).

FIG. 4 shows the results of the in vivo screen of 16 mouse specific(AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs in C57/BL6 mice (5animals/group). Total serum cholesterol levels were averaged over eachtreatment group and compared to a control group treated with PBS or acontrol group treated with an unrelated siRNA (blood coagulation factorVII).

FIG. 5 shows the results of the in viva screen of 16 human/mouse/ratcrossreactive (AL-DP-9311 through AL-DP-9326) PCSK9 siRNAs in C57/BL6mice (5 animals/group). Total serum cholesterol levels were averagedover each treatment group and compared to a control group treated withPBS or a control group treated with an unrelated siRNA (bloodcoagulation factor VII).

FIG. 6 shows a comparison of the in vitro and in vivo results forsilencing PCSK9.

FIG. 7A and FIG. 7B show in vitro results for silencing PCSK9 usingmonkey primary hepatocytes.

FIG. 8 shows in vivo activity of LNP-01 formulated siRNAs to pcsk-9.

FIG. 9 shows in vivo activity of LNP-01 Formulated chemically modified9314 and 10792 parent molecules at different times. Clearly modifiedversions of 10792 display in vivo silencing activity.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a solution to the problem of treating diseasesthat can be modulated by the down regulation of the PCSK9 gene, by usingdouble-stranded ribonucleic acid (dsRNA) to silence the PCSK9 gene thusproviding treatment for diseases such as hyperlipidemia.

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the PCSK9gene in a cell or mammal using the dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases that can be modulated by down regulating the expression of thePCSK9 gene dsRNA directs the sequence-specific degradation of mRNAthrough a process known as RNA interference (RNAi).

The dsRNA of die invention comprises an RNA strand (the antisensestrand) having a region which is less than 30 nucleotides in length,generally 19-24 nucleotides in length, and is substantiallycomplementary to at least past of an mRNA transcript of the FCSK9 gene.The use of these dsRNAs enables the targeted degradation of an mRNA thatis involved in sodium transport. Using cell-based and animal assays, thepresent inventors have demonstrated that very low dosages of these dsRNAcan specifically and efficiently mediate RNAi, resulting in significantinhibition of expression of the PCSK9 gene. Thus, the methods andcompositions of the invention comprising these dsRNAs are useful fortreating pathological processes which can be mediated by down regulatingPCSK9, such as in the treatment of hyperlipidemia.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of thetarget FCSK9 gene, as well as compositions and methods for treatingdiseases that can be modulated by down regulating the expression ofPCSK9, such as hyperlipidemia. The pharmaceutical compositions of theinvention comprise a dsRNA having an antisense strand comprising aregion of complementarity which is less than 30 nucleotides in length,generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an RNA transcript of the PCSK9 gene,together with a pharmaceutically acceptable carrier.

Accordingly, certain aspects of the invention provide pharmaceuticalcompositions comprising the dsRNA of the invention together with apharmaceutically acceptable carrier, methods of using the compositionsto inhibit expression of the PCSK9 gene, and methods of using thepharmaceutical compositions to treat diseases that can be modulated bydown regulating the expression of PCSK9.

I. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, and uracil may be replacedby other moieties without substantially altering the base pairingproperties of an oligonucleotide comprising a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.

As used herein, “PCSK9” refers to the proprotein convertase subtilisinkexin 9 gene or protein (also known as FIB, HCHOLA3, NARC-1, NARC1),mRNA sequences to PCSK9 are provided as human: NM_(—)174936; mouse:NM_(—)153565, and rat: NM_(—)199253.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the PCSK9 gene, including mRNA that is a product of RNA processing ofa primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding PCSK9). For example, a polynucleotide iscomplementary to at least a part of a PCSK9 mRNA if the sequence issubstantially complementary to a non-interrupted portion of a mRNAencoding PCSK9.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. The two strands forming the duplexstructure may be different portions of one larger RNA molecule, or theymay be separate RNA molecules. Where separate RNA molecules, such dsRNAare often referred to in the literature as siRNA (“short interferingRNA”). Where the two strands are part of one larger molecule, andtherefore are connected by an uninterrupted chain, of nucleotidesbetween the 3′-end of one strand and the 5′end of the respective otherstrand forming the duplex structure, the connecting RNA chain isreferred to as a “hairpin loop”, “short hairpin RNA” or “shRNA”. Wherethe two strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker”. The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA minus any overhangs that are present in the duplex. Inaddition to the duplex structure, a dsRNA may comprise one or morenucleotide overhangs. In addition, as used in this specification,“dsRNA” may include chemical modifications to ribonucleotides, includingsubstantial modifications at multiple nucleotides and including alltypes of modifications disclosed herein or known in the art. Any suchmodifications, as used in an siRNA type molecule, are encompassed by“dsRNA” for the purposes of this specification and claims.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides drat protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt, ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule. For clarity, chemical caps or non-nucleotidechemical moieties conjugated to the 3′ end or 5′ end of an siRNA are notconsidered in determining whether an siRNA has an overhang or is bluntended.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of”, in as far as theyrefer to the PCSK9 gene, herein refer to the at least partial,suppression of the expression of the PCSK9 gene, as manifested by areduction of the amount of mRNA-transcribed from the PCSK9 gene whichmay be isolated from a first cell or group of cells in which the PCSK9gene is transcribed and which has or have been treated such that theexpression of the PCSK9 gene is inhibited, as compared to a second cellor group of cells substantially identical to the first cell or group ofcells but which has or have not been so treated (control cells). Thedegree of inhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to PCSK9 genetranscription, e.g. the amount of protein encoded by the PCSK9 genewhich is secreted by a cell, or the number of cells displaying a certainphenotype, e.g. apoptosis. In principle, PCSK9 gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given dsRNA inhibitsthe expression of the PCSK9 gene by a certain, degree and therefore isencompassed by the instant invention, the assay provided in the Examplesbelow shall serve as such reference.

For example, in certain instances, expression of the PCSK9 gene issuppressed by at least about 20%, 25%, 35%, or 50% by administration ofthe double-stranded oligonucleotide of the invention, in someembodiment, the PCSK9 gene is suppressed by at least about 60%, 70%, or80% by administration of the double-stranded oligonucleotide of theinvention. In some embodiments, the PCSK9 gene is suppressed by at leastabout 85%, 90%, or 95% by administration of the double-strandedoligonucleotide of the invention. Tables 1, 2, provides a wide range ofvalues for inhibition of expression obtained in an in vitro assay usingvarious PCSK9 dsRNA molecules at various concentrations.

As used herein in the context of PCSK9 expression, the terms “treat”,“treatment”, and the like, refer to relief from or alleviation ofpathological processes which, can be mediated by down regulating PCSK9gene. In the context of the present invention insofar as it relates toany of the other conditions recited herein below (other thanpathological processes which can be mediated by down regulating thePCSK9 gene), the terms “treat”, “treatment”, and the like mean torelieve or alleviate at least one symptom associated with suchcondition, or to slow or reverse the progression of such condition. Forexample, in the context of hyperlipidemia, treatment will involve adecrease in serum lipid levels.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment prevention, or management ofpathological processes which can be mediated by down regulating thePCSK9 gene on or an overt symptom of pathological processes which can bemediated by down regulating the PCSK9 gene. The specific amount that istherapeutically effective can be readily determined by ordinary medicalpractitioner, and may vary depending on factors known in the art, suchas, e.g. the type of pathological processes which, can be mediated bydown regulating the PCSK9 gene, the patient's history and age, the stageof pathological processes which can be mediated by down regulating PCSK9gene expression, and the administration of other anti-pathologicalprocesses which can be mediated by down regulating PCSK9 geneexpression.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof and are described in more detailbelow. The term specifically excludes cell culture medium.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. Double-strand Ribonucleic Acid (dsRNA)

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the PCSK9 genein a cell or mammal, wherein the dsRNA comprises an antisense strandcomprising a region of complementarity which is complementary to atleast a part of an mRNA formed in the expression of the PCSK9 gene, andwherein the region of complementarity is less than 30 nucleotides inlength, generally 19-24 nucleotides in length, and wherein said dsRNA,upon contact with a cell expressing said PCSK9 gene, inhibits theexpression of said PCSK9 gene by at least 40%. The dsRNA comprises twoRNA strands that are sufficiently complementary to hybridize to form aduplex structure. One strand of the dsRNA (the antisense strand)comprises a region of complementarity that is substantiallycomplementary, and generally fully complementary, to a target sequence,derived from the sequence of an mRNA formed during the expression of thePCSK9 gene, the other strand (the sense strand) comprises a region whichis complementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. Generally, the duplex structure is between 15 and 30, moregenerally between 18 and 25, yet more generally between 19 and 24, andmost generally between 19 and 21 base pairs in length. Similarly, theregion of complementarity to the target sequence is between 15 and 30,more generally between 18 and 25, yet more generally between 19 and 24,and most generally between 19 and 21 nucleotides in length. The dsRNA ofthe invention may further comprise one or more single-strandednucleotide overhang(s). The dsRNA can be synthesized by standard methodsknown in the art as further discussed, below, e.g., by use of anautomated DNA synthesizer, such as are commercially available from, forexample, Biosearch, Applied Biosystems, Inc. In a preferred embodiment,the PCSK9 gene is the human PCSK9 gene. In specific embodiments, theantisense strand of the dsRNA comprises a strand selected from the sensesequences of Tables 1 and 2, and a second sequence selected from thegroup consisting of the antisense sequences of Tables 1 and 2.Alternative antisense agents that target, elsewhere in the targetsequence provided in Tables 1 and 2, can readily be determined using thetarget sequence and the flanking PCSK9 sequence.

in further embodiments, the dsRNA comprises at least one nucleotidesequence selected from the groups of sequences provided in Tables 1 and2 in other embodiments, the dsRNA comprises at least two sequencesselected from this group, wherein one of the at least two sequences iscomplementary to another of the at least two sequences, and one of theat least two sequences is substantially complementary to a sequence ofah mRNA generated in the expression of the PCSK9 gene. Generally, thedsRNA comprises two oligonucleotides, wherein one oligonucleotide isdescribed as the sense strand in Tables 1 and 2 and the secondoligonucleotide is described as the antisense strand in Tables 1 and 2

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well, in theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 1 and 2, the dsRNAs of theinvention can comprise at least one strand of a length, of minimally 21nt. It can be reasonably expected that shorter dsRNAs comprising one ofthe sequences of Tables 1 and 2 minus only a few nucleotides on one orboth, ends may be similarly effective as compared to the dsRNAsdescribed above. Hence, dsRNAs comprising a partial sequence of at least15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of thesequences of Tables 1 and 2, and differing in their ability to inhibitthe expression of the PCSK9 gene in a FACS assay as described hereinbelow by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNAcomprising the full sequence, are contemplated by the invention. FurtherdsRNAs that cleave within the target sequence provided in Tables 1 and 2can readily be made using the PCSK9 sequence and the target sequenceprovided.

In addition, the RNAi agents provided in Tables 1 and 2 identify a sitein the PCSK9 mRNA that is susceptible to RNAi based cleavage. As suchthe present invention further includes RNAi agents that target, withinthe sequence targeted by one of the agents of the present invention. Asused herein a second RNAi agent is said to target within the sequence ofa first RNAi agent if the second RNAi agent cleaves the message anywherewithin the mRNA that is complementary to the antisense strand of thefirst RNAi agent. Such a second agent will generally consist of at least15 contiguous nucleotides from one of the sequences provided in Tables 1and 2 coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in the PCSK9 gene. For example, thelast 15 nucleotides of SEQ ID NO:1 (minus the added AA sequences)combined with the next 6 nucleotides from the target PCSK9 gene producesa single strand agent of 21 nucleotides that is based on one of thesequences provided in Tables 1 and 2.

The dsRNA of the invention, can contain one or more mismatches to thetarget sequence. In a preferred embodiment, the dsRNA of the inventioncontains no more than 3 mismatches. If the antisense strand of the dsRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of the PCSK9 gene, the dsRNA generally doesnot contain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of the PCSK9 gene. Consideration of the efficacy ofdsRNAs with mismatches in inhibiting expression of the PCSK9 gene isimportant, especially if the particular region of complementarity in thePCSK9 gene is known to have polymorphic sequence variation within thepopulation.

In one embodiment at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. Moreover, thepresent inventors have discovered that the presence of only onenucleotide overhang strengthens the interference activity of the dsRNA,without affecting its overall stability, dsRNA having only one overhanghas proven particularly stable and effective in vivo, as well as in avariety of cells, cell culture mediums, blood, and serum. Generally, thesingle-stranded overhang is located, at the 3′-terminal end of theantisense strand or, alternatively, at the 3′-terminal end of the sensestrand. The dsRNA may also have a blunt end, generally located at the5′-end of the antisense strand. Such dsRNAs have improved stability andinhibitory activity, thus allowing administration at low dosages, i.e.,less than 5 mg/kg body weight of the recipient per day. Generally, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids of die invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry”. Beaucage, S. L. et.al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which ishereby incorporated herein by reference. Chemical modifications mayinclude, but are not limited to 2′ modifications, modifications at othersites of the sugar or base of an oligonucleotide, introduction ofnon-natural bases into the olibonucleotide chain, covalent attachment toa ligand or chemical moiety, and replacement of internucleotidephosphate linkages with alternate linkages such as thiophosphates. Morethan one such modification may be employed.

Chemical linking of the two separate dsRNA strands may be achieved byany of a variety of well-known techniques, for example by introducingcovalent, ionic or hydrogen bonds; hydrophobic interactions, van derWaals or stacking interactions; by means of metal-ion coordination, orthrough, use of purine analogues. Generally, the chemical groups thatcan be used to modify the dsRNA include, without limitation, methyleneblue; bifunctional groups, generally bis-(2-chloroethyl)amine;N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. Inone embodiment, the linker is a hexa-ethylene glycol linker. In thiscase, the dsRNA are produced by solid phase synthesis and thehexa-ethylene glycol linker is incorporated according to standardmethods (e.g., Williams, D. J., and K. B. Hail, Biochem. (1996)35:14665-14670). In a particular embodiment, the 5′-end of the antisensestrand and the 3′-end of the sense strand are chemically linked via ahexaethylene glycol linker. In another embodiment, at least onenucleotide of the dsRNA comprises a phosphorothioate orphosphorodithioate groups. The chemical bond at the ends of the dsRNA isgenerally formed by triple-helix bonds. Tables 1 and 2 provides examplesof modified RNAi agents of the invention.

In yet another embodiment, the nucleotides at one or both of the twosingle strands may be modified to prevent or inhibit the degradationactivities of cellular enzymes, such, as, for example, withoutlimitation, certain nucleases. Techniques for inhibiting the degradationactivity of cellular enzymes against nucleic acids are known in the artincluding, but not limited to, 2′-amino modifications, 2′-amino sugarmodifications, 2′-F sugar modifications, 2′-F modifications, 2′-alkylsugar modifications, uncharged backbone modifications, morpholinomodifications, 2′-methyl modifications, and phosphoramidate (sec, e.g.,Wagner, Nat. Med. (1995) 1:1116-8). Thus, at least one 2′-hydroxyl groupof the nucleotides on a dsRNA is replaced by a chemical group, generallyby a 2′-amino or a 2′-methyl group. Also, at least one nucleotide may bemodified to form a locked, nucleotide. Such locked nucleotide contains amethylene bridge that connects the 2′-oxygen of ribose with the4′-carbon of ribose. Oligonucleotides containing the locked nucleotideare described in Koshkin, A. A., et al., Tetrahedron (1998), 54:3607-3630) and Obika, S. et al. Tetrahedron Lett. (1998), 39;5401-5404). Introduction of a locked nucleotide into an oligonucleotideimproves the affinity for complementary sequences and increases themelting temperature by several degrees (Braasch, D. A. and D. R. Corey,Chem. Biol. (2001), 8:1-7).

Conjugating a ligand to a dsRNA can enhance its cellular absorption aswell as targeting to a particular tissue or uptake by specific types ofcells such as liver cells. In certain instances, a hydrophobic ligand isconjugated to the dsRNA to facilitate direct permeation of the cellularmembrane and or uptake across the liver cells. Alternatively, the ligandconjugated, to the dsRNA is a substrate for receptor-mediatedendocytosis. These approaches have been used to facilitate cellpermeation of antisense oligonucleotides as well as dsRNA agents. Forexample, cholesterol has been conjugated to various antisenseoligonucleotides resulting in compounds that are substantially moreactive compared to their non-conjugated analogs. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103. Otherlipophilic compounds that have been conjugated to oligonucleotidesinclude 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, andmenthol. One example of a ligand for receptor-mediated endocytosis isfolic acid. Folic acid enters the cell by folate-receptor-mediatedendocytosis. dsRNA compounds bearing folic acid would be efficientlytransported into the cell via the folate-receptor-mediated endocytosis.Li and coworkers report that attachment of folic acid to the 3′-terminusof an oligonucleotide resulted in an 8-fold increase in cellular uptakeof the oligonucleotide. Li, S.; Deshmukh, H, M.; Huang, L. Pharm. Res.1998, 15, 1540. Other ligands that have been conjugated tooligonucleotides include polyethylene glycols, carbohydrate clusters,cross-linking agents, porphyrin conjugates, delivery peptides and lipidssuch as cholesterol.

In certain instances, conjugation of a cationic ligand tooligonucleotides results in improved resistance to nucleases.Representative examples of cationic ligands are propylammonium anddimethylpropylammonium. Interestingly, antisense oligonucleotides werereported to retain their high binding affinity to mRNA when the cationicligand was dispersed throughout the oligonucleotide. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103 and referencestherein.

The ligand-conjugated dsRNA of the invention may be synthesized by theuse of a dsRNA that bears a pendant reactive functionality, such as thatderived from the attachment of a linking molecule onto the dsRNA. Thisreactive oligonucleotide may be reacted directly withcommercially-available ligands, ligands that are synthesized bearing anyof a variety of protecting groups, or ligands that have a linking moietyattached thereto. The methods of the invention facilitate the synthesisof ligand-conjugated dsRNA by the use of, in some preferred embodiments,nucleoside monomers that have been appropriately conjugated with ligandsand that may further be attached to a solid-support material. Suchligand-nucleoside conjugates, optionally attached to a solid-supportmaterial, are prepared according to some preferred embodiments of themethods of the invention via reaction of a selected serum-binding ligandwith a linking moiety located on the 5′ position of a nucleoside oroligonucleotide. In certain instances, an dsRNA bearing an aralkylligand attached to the 3′-terminus of the dsRNA is prepared by firstcovalently attaching a monomer building block to a controlled-pore-glasssupport via a long-chain aminoalkyl group. Then, nucleotides are bondedvia standard solid-phase synthesis techniques to the monomerbuilding-block bound to the solid support. The monomer building blockmay be a nucleoside or other organic compound that is compatible withsolid-phase synthesis.

The dsRNA used in the conjugates of the invention may be convenientlyand routinely made through the well-known technique of solid-phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is also known to use similar techniques toprepare other oligonucleotides, such as the phosphorothioates andalkylated derivatives.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. Pat. Nos. 5,138,045and 5,218,105, drawn to polyamine conjugated oligonucleotides: U.S. Pat.No. 5,212,295, drawn to monomers for the preparation of oligonucleotideshaving chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and5,541,307, drawn to oligonucleotides having modified backbones; U.S.Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and thepreparation thereof through reductive coupling; U.S. Pat. No. 5,457,191,drawn to modified nucleobases based on the 3-deazapurine ring system andmethods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides havingβ-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups may be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2′-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.Nos. 5,223,168, and 5,608,046, both drawn to conjugated 4′-desmethylnucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn tobackbone-modified oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and5,459,255, drawn to, inter alia, methods of synthesizing2′-fluoro-oligonucleotides.

In the ligand-conjugated dsRNA and ligand-molecule bearingsequence-specific linked nucleosides of the invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already hear a linkingmoiety, die synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide.Oligonucleotide conjugates bearing a variety of molecules such assteroids, vitamins, lipids aid reporter molecules, has previously beendescribed (see Manoharan et al. PCT Application WO 93/07883). In apreferred embodiment, the oligonucleotides or linked nucleosides of theinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

The incorporation of a 2′-O-methyl 2′-O-ethyl, 2′-O-propyl 2′-O-allyl,2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of anoligonucleotide confers enhanced hybridization properties to theoligonucleotide. Further, oligonucleotides containing phosphorothioatebackbones have enhanced nuclease stability. Thus, functionalized, linkednucleosides of the invention can be augmented to include either or botha phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-O-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group. A summarylisting of some of the oligonucleotide modifications known in the art isfound at, for example, PCT Publication WO 200370918.

In some embodiments, functionalized nucleoside sequences of theinvention possessing an amino group at the 5′-terminus are preparedusing a DNA synthesizer, and then reacted with an active esterderivative of a selected ligand. Active ester derivatives are well knownto those skilled in the art. Representative active esters includeN-hydroxsuccinimide esters, tetrafluorophenolic esters,pentafluorophenolic esters and pentachlorophenolic esters. The reactionof the amino group and the active ester produces an oligonucleotide inwhich the selected ligand is attached to the 5′-position through alinking group. The amino group at the 5′-terminus can be preparedutilizing a 5′-Amino-Modifier C6 reagent. In one embodiment, ligandmolecules may be conjugated to oligonucleotides at the 5′-position bythe use of a ligand-nucleoside phosphoramidite wherein the ligand islinked to the 5′-hydroxy group directly or indirectly via a linker. Suchligand-nucleoside phosphoramidites are typically used at the end of anautomated synthesis procedure to provide a ligand-conjugatedoligonucleotide bearing the ligand at the 5′-terminus.

Examples of modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acidforms are also included.

Representative United States patents relating to the preparation of theabove phosphorus-atom-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is hereinincorporated by reference.

Examples of modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleosides) havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents relating to the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 1264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In certain instances, the oligonucleotide may be modified by anon-ligand group. A number of non-ligand molecules have been conjugatedto oligonucleotides in order to enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide, and proceduresfor performing such conjugations are available in the scientificliterature. Such non-ligand moieties have included lipid moieties, suchas cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994,4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann.N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem.Let, 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov etal., FEBS Lett, 1990, 259:327; Svinarchuk et al., Biochimie, 1993,75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al. Tetrahedron Lett., 1995, 36:3651; Shea et al.,Noel.Acids Res., 1990, 18:3777), a poly amine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al. Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al. Biochim. Biophys. Acta, 1995,1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterolmoiety (Crooks et al., J. Pharmacol Exp. Ther., 1996, 277:923).Representative United States patents that teach the preparation of sucholigonucleotide conjugates have been listed above. Typical conjugationprotocols involve the synthesis of oligonucleotides bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the oligonucleotide still bound to the solidsupport or following cleavage of the oligonucleotide in solution phase.Purification of the oligonucleotide conjugate by HPLC typically affordsthe pure conjugate. The use of a cholesterol conjugate is particularlypreferred since such a moiety can increase targeting liver cells, a siteof PCSK9 expression.

Vector Encoded RNAi Agents

The dsRNA of the invention can also be expressed from recombinant viralvectors intracellularly in vivo. The recombinant viral vectors of theinvention comprise sequences encoding the dsRNA of the invention and anysuitable promoter for expressing the dsRNA sequences. Suitable promotersinclude, for example, the U6 or H1 RNA pol III promoter sequences andthe cytomegalovirus promoter. Selection of other suitable promoters iswithin the skill in the art. The recombinant viral vectors of theinvention can also comprise inducible or regulatable promoters forexpression of the dsRNA in a particular tissue or in a particularintracellular environment. The use of recombinant viral vectors todeliver dsRNA of the invention to cells in vivo is discussed in moredetail below.

dsRNA of the invention can be expressed from a recombinant viral vectoreither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g.,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorswhich express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801,the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392; 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the dsRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector comprising, for example, either the U6 orH1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a methodfor constructing the recombinant AV vector, and a method for deliveringthe vector into target cells, are described in Xia H et al. (2002), Nat.Biotech, 20; 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.Nos. 5,252,479; 5,139,941; International Patent Application No. WO94/13788; and international Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

III. Pharmaceutical Compositions Comprising dsRNA

In one embodiment, the invention provides pharmaceutical compositionscomprising a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition comprising the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of the PCSK9 gene, such as pathological processeswhich, can be mediated by down regulating PCSK9 gene expression, such ashyperlipidemia. Such pharmaceutical compositions are formulated, basedon the mode of delivery. One example is compositions that are formulatedfor delivery to the liver via parenteral delivery.

The pharmaceutical compositions of the invention are administered indosages sufficient to inhibit expression of the PCSK9 gene. The presentinventors have found that, because of their improved efficiency,compositions comprising the dsRNA of the invention can be administeredat surprisingly low dosages. A dosage of 5 mg dsRNA per kilogram bodyweight of recipient per day is sufficient to inhibit or suppressexpression of the PCSK9 gene and may be administered systematically tothe patient.

In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0milligrams per kilogram body weight of the recipient per day, generallyin the range of 1 microgram to 1 mg per kilogram body weight per day.The pharmaceutical composition may be administered once daily, or thedsRNA may be administered as two, three, or more sub-closes atappropriate intervals throughout the day or even using continuousinfusion or delivery through a controlled release formulation. In thatcase, the dsRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage. The dosage unit canalso the compounded for delivery over several days, e.g., using aconventional sustained release formulation which provides sustainedrelease of the dsRNA over a several day period. Sustained releaseformulations are well known in the art.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processeswhich can be mediated by down regulating PCSK9 gene expression. Suchmodels are used for in vivo testing of dsRNA, as well as for determininga therapeutically effective dose.

Any method can be used to administer a dsRNA of the present invention toa mammal. For example, administration can be direct; oral; or parenteral(e.g., by subcutaneous, intraventricular, intramuscular, orintraperitoneal injection, or by intravenous drip). Administration canbe rapid (e.g., by injection), or can occur over a period of time (e.g.,by slow infusion or administration of slow release formulations).

Typically, when treating a mammal with hyperlipidemia, the dsRNAmolecules are administered systemically via parental means. For example,dsRNAs, conjugated or unconjugate or formulated with or withoutliposomes, can be administered intravenously to a patient. For such, adsRNA molecule can be formulated into compositions such as sterile andnon-sterile aqueous solutions, non-aqueous solutions in common solventssuch as alcohols, or solutions in liquid or solid oil bases. Suchsolutions also can contain buffers, diluents, and other suitableadditives. For parenteral, intrathecal, or intraventricularadministration, a dsRNA molecule can be formulated into compositionssuch as sterile aqueous solutions, which also can contain buffers,diluents, and other suitable additives (e.g., penetration enhancers,carrier compounds, and other pharmaceutically acceptable carriers).

In addition, dsRNA molecules can be administered to a mammal as biologicor abiologic means as described in, for example, U.S. Pat. No.6,271,359, Abiologic delivery can be accomplished by a variety ofmethods including, without limitation, (1) loading liposomes with adsRNA acid molecule provided herein and (2) complexing a dsRNA moleculewith lipids or liposomes to form nucleic acid-lipid or nucleicacid-liposome complexes. The liposome can be composed of cationic aidneutral lipids commonly used to transfect cells in vitro. Cationiclipids can complex (e.g., charge-associate) with negatively chargednucleic acids to form liposomes. Examples of cationic liposomes include,without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP.Procedures for forming liposomes are well, known in the art. Liposomecompositions can be formed, for example, from phosphatidylcholine,dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine,dimyristoyl phosphatidyl glycerol or dioleoyl phosphatidylethanolamine.Numerous lipophilic agents are commercially available, includingLipofectin®, (Invitrogen/Life Technologies, Carlsbad, Calif.) andEffectene™, (Qiagen, Valencia, Calif.). In addition, systemic deliverymethods can be optimized using commercially available cationic lipidssuch as DDAB or DOTAP, each of which can be mixed with a neutral lipidsuch as DOPE or cholesterol. In some cases, liposomes such as thosedescribed by Templeton et al. (Nature Biotechnology, 15: 647-652 (1997))can be used. In other embodiments, polycations such as polyethyleneiminecan be used to achieve delivery in vivo and ex vivo (Boletta et al., J.Am. Soc. Nephrol. 7: 1728 (1996)). Additional information regarding theuse of liposomes to deliver nucleic acids can be found in U.S. Pat. No.6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al. 2005,Nat Biotechnol. 23(8): 1002-7.

Biologic delivery can be accomplished by a variety of methods including,without limitation, the use of viral vectors. For example, viral vectors(e.g., adenovirus and herpesvirus vectors) can be used to deliver dsRNAmolecules to liver cells. Standard molecular biology techniques can beused to introduce one or more of the dsRNAs provided herein into one ofthe many different viral vectors previously developed to deliver nucleicacid to cells. These resulting viral vectors can be used to deliver theone or more dsRNAs to cells by, for example, infection.

dsRNAs of the present invention can be formulated in a pharmaceuticallyacceptable carrier or diluent. A “pharmaceutically acceptable carrier”(also referred to herein as an “excipient”) is a pharmaceuticallyacceptable solvent, suspending agent, or any other pharmacologicallyinert vehicle. Pharmaceutically acceptable carriers can be liquid orsolid, and can be selected with the planned manner of administration inmind so as to provide for the desired bulk, consistency, and otherpertinent transport and chemical properties. Typical pharmaceuticallyacceptable carriers include, by way of example and not limitation;water; saline solution; binding agents (e.g., polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars,gelatin, or calcium sulfate); lubricants (e.g., starch, polyethyleneglycol, or sodium acetate); disintegrates (e.g., starch or sodium starchglycolate); and wetting agents (e.g., sodium, lauryl sulfate).

In addition, dsRNA that target the PCSK9 gene can be formulated intocompositions containing the dsRNA admixed, encapsulated, conjugated, orotherwise associated with other molecules, molecular structures, ormixtures of nucleic acids. For example, a composition containing one ormore dsRNA agents that target the PCSK9 gene can contain othertherapeutic agents such as other lipid lowering agents (e.g., statins).

Methods for Treading Diseases that can be Modulated by Down Regulatingthe Expression of PCSK9

The methods and compositions described herein can be used to treatdiseases and conditions that can be modulated by down regulating PCSK9gene expression. For example, the compositions described herein can beused to treat hyperlipidemia and other forms of lipid inbalance such ashypercholesterolemia, hypertriglyceridemia and the pathologicalconditions associated with these disorders such as heart and circulatorydiseases.

Methods for Inhibiting Expression of the PCSK9 Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of the PCSK9 gene in a mammal. The method comprisesadministering a composition of the invention to the mammal such thatexpression of the target PCSK9 gene is silenced. Because of their highspecificity, the dsRNAs of the invention specifically target RNAs(primary or processed) of the target PCSK9 gene. Compositions andmethods for inhibiting the expression of these PCSK9 genes using dsRNAscan be performed as described elsewhere herein.

In one embodiment, the method comprises administering a compositioncomprising a dsRNA, wherein the dsRNA comprises a nucleotide sequencewhich is complementary to at least a part of an RNA transcript of thePCSK9 gene of the mammal to be treated. When the organism to be treatedis a mammal such as a human, the composition may be administered by anymeans known in the art including, but not limited to oral or parenteralroutes, including intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol) administration. In preferred embodiments, thecompositions are administered by intravenous infusion or injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. Ail publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control, in addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES

Gene Walking of the PCSK9 Gene

siRNA design was carried out to identity in two separate selections

a) siRNAs targeting PCSK9 human and either mouse or rat mRNA and

b) all human reactive siRNAs with predicted specificity to the targetgene PCSK9.

mRNA sequences to human, mouse and rat PCSK9 were used: Human sequenceNM_(—)174936.2 was used as reference sequence during the complete siRNAselection procedure.

19 mer stretches conserved in human and mouse, and human and rat PCSK9mRNA sequences were identified in the first step, resulting in theselection of siRNAs crossreactive to human and mouse, and siRNAscrossreactive to human and rat targets

SiRNAs specifically targeting human. PCSK9 were identified in a secondselection. All potential 19mer sequences of human PCSK9 were extractedand defined as candidate target sequences. Sequences cross-reactive tohuman, monkey, and those cross-reactive to mouse, rat, human and monkeyare all listed in Tables 1 and 2. Chemically modified versions of thosesequences and their activity in both in vitro and in vivo assays arealso listed in tables 1 and 2 and examples given in FIGS. 2-8.

In order to rank candidate target sequences and their correspondingsiRNAs and select appropriate ones, their predicted potential forinteracting with irrelevant targets (off-target potential) was taken asa ranking parameter. siRNAs with low off-target potential were definedas preferable and assumed to be more specific in vivo.

For predicting siRNA-specific off-target potential, the followingassumptions were made;

1) positions 2 to 9 (counting 5′ to 3′) of a strand (seed region) maycontribute more to off-target potential than rest of sequence (non-seed,and cleavage site region)

2) positions 10 and 11 (counting 5′ to 3′) of a strand (cleavage siteregion) may contribute more to off-target potential than non-seed region

3) positions 1 and 19 of each strand are not relevant for off-targetinteractions

4) an off-target score can be calculated for each gene and each strand,based on complementarity of siRNA strand sequence to the gene's sequenceand position of mismatches

5) number of predicted off-targets as well as highest off-target scoremust be considered for off-target potential

6) off-tar get scores are to be considered more relevant for off-targetpotential than numbers of off-targets

7) assuming potential abortion of sense strand activity by internalmodifications introduced, only off-target potential of antisense strandwill be relevant

To identify potential off-target genes, 19mer candidate sequences weresubjected to a homology search against publically available human mRNAsequences.

The following off-target properties for each 19mer input sequence wereextracted for each off-target gene to calculate the off-target score:

Number of mismatches in non-seed region

Number of mismatches in seed region

Number of mismatches in cleavage site region

The off-target score was calculated for considering assumption 1 to 3 asfollows:

Off-target score=number of seed mismatches*10

-   -   number of cleavage site mismatches*1.2    -   number of non-seed mismatches*1

The most relevant off-target gene for each siRNA corresponding to theinput 19mer sequence was defined as the gene with the lowest off-targetscore. Accordingly, the lowest off-target score was defined as therelevant off-target score for each siRNA.

dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These, building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent, protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried, out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength, of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany),Double stranded RNA was generated, by mixing an equimolar solution ofcomplementary strands, in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), healed in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol-3′), an appropriately modified solid support was used forRNA synthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature untilcompletion of the reaction was ascertained by TLC. After 19 h thesolution was partitioned with dichloromethane (3×100 mL). The organiclayer was dried with anhydrous sodium sulfate, filtered and evaporated.The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimide(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.Completion of the reaction was ascertained by TLC. The reaction mixturewas concentrated under vacuum and ethyl acetate was added to precipitatediisopropyl urea. The suspension was filtered. The filtrate was washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer was dried over sodium sulfate and concentrated togive the crude product which was purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated under vacuum, water was addedto the residue, and the product was extracted with ethyl acetate. Thecrude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 mL of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water. Theresultant mixture was extracted twice with 100 mL of dichloromethaneeach and the combined organic extracts were washed twice with 10 mL ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 mL of toluene, cooled to 0° C. and extracted with three50 mL portions of cold phi 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40mL portions of chloroform which were combined, dried and evaporated todryness. The residue was purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyelopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) was added, the mixture was extracted with ethylacetate (3×40mL). The combined ethylacetate layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxy-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was earned out at room temperature overnight. Thereaction was quenched by the addition of methanol. The reaction mixturewas concentrated under vacuum and to the residue dichloromethane (50 mL)was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

Cholesterol derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMA (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane. (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution, was agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The achieved loading of die CPG was measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”.) was performed as described inWO 2004/065601, except that, for the cholesteryl derivative, theoxidation step was performed using the Beaucage reagent in order tointroduce a phosphorothioate linkage at the 5′-end of the nucleic acidoligomer.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 1-2.

TABLE 1-2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation^(a) Nucleotide(s) A, a2′-deoxy-adenosine-5′-phosphate, adenosine-5′- phosphate C, c2′-deoxy-cytidine-5′-phosphate, cytidine-5′- phosphate G, g2′-deoxy-guanosine-5′-phosphate, guanosine-5′- phosphate T, t2′-deoxy-thymidine-5′-phosphate, thymidine-5′- phosphate U, u2′-deoxy-uridine-5′-phosphate, uridine-5′- phosphate N, n any2′-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c or u) Am2′-O-methyladenosine-5′-phosphate Cm 2′-O-methylcytidine-5′-phosphate Gm2′-O-methylguanosine-5′-phosphate Tm 2′-O-methyl-thymidine-5′-phosphateUm 2′-O-methyluridine-5′-phosphate Af2′-fluoro-2′-deoxy-adenosine-5′-phosphate Cf2′-fluoro-2′-deoxy-cytidine-5′-phosphate Gf2′-fluoro-2′-deoxy-guanosine-5′-phosphate Tf2′-fluoro-2′-deoxy-thymidine-5′-phosphate Uf2′-fluoro-2′-deoxy-uridine-5′-phosphate A, C, G, T, U, a, underlined:nucleoside-5′-phosphorothioate c, g, t, u am, cm, gm, tm, underlined:2-O-methyl-nucleoside-5′-phosphorothioate um ^(a)capital lettersrepreseat 2′-deoxyribonucleotides (DNA), lower case letters representribonucleotides (RNA)

PCSK9 siRNA Screening in HuH7, HepG2, Hela and Primary MonkeyHepatocytes Discovers Highly Active Sequences

HuH-7 cells were obtained from JCRB Cell Bank (Japanese Collection ofResearch Bioresources) (Shinjuku, Japan, cat. No.: JCRB0403) Cells werecultured in Dulbecco's MEM (Biochrom AG, Berlin, Germany, eat. No.F0435) supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG,Berlin, Germany, cat. No. S0115), Penicillin 100 U/ml, Streptomycin 100μg/ml (Biochrom AG, Berlin, Germany, cat. No, A2213) and 2 mM L-Glutamin(Biochrom AG, Berlin, Germany, cat. No K0282) at 37° C. in an atmospherewith 5% CO₂ in a humidified incubator (Heraeus HERAcell, KendroLaboratory Products, Langenselbold, Germany). HepG2 and Hela cells wereobtained from American Type Culture Collection (Rockville, Md., cat. No,HB-8065) and cultured in MEM (Gibco Invitrogen, Karlsruhe, Germany, eat.No, 21090-022) supplemented to contain 10% fetal calf serum (FCS)(Biochrom AG, Berlin, Germany, cat. No. S0115), Penicillin 100 U/ml,Streptomycin 100 μg/ml (Biochrom AG, Berlin, Germany, cat. No. A2213),1× Non Essential Amino Acids (Biochrom AG, Berlin, Germany, cat. No.K-0293), and 1 mM Sodium Pyruvate (Biochrom AG, Berlin, Germany, cat.No. L-0473) at 37° C. in an atmosphere with 5% CO₂ in a humidifiedincubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold,Germany).

For transfection with siRNA, HuH7, HepG2, or Hela cells were seeded at adensity of 2.0×10⁴ cells/well in 96-well plates and transfecteddirectly. Transfection of siRNA (30 nM for single dose screen) wascarried out with lipofectamine 2000 (Invitrogen GmbH, Karlsruhe,Germany, cat. No. 11668-019) as described by the manufacturer.

24 hours after transfection HuH7 and HepG2 cells were lysed and PCSK9mRNA levels were quantified with the Quantigene Explore Kit(Genosprectra, Dumbarton Circle Fremont, USA, cat. No. QG-000-02)according to the protocol. PCSK9 mRNA levels were normalized to GAP-DHmRNA. For each siRNA eight individual datapoints were collected, siRNAduplexes unrelated to PCSK9 gene were used as control. The activity of agiven. PCSK9 specific siRNA duplex was expressed as percent PCSK9 mRNAconcentration in treated cells relative to PCSK9 mRNA concentration incells treated with the control siRNA duplex.

Primary cynomolgus monkey hepatocytes (cryopreserved) were obtained fromIn vitro Technologies, Inc. (Baltimore, Md., USA, cat No M00305) andcultured in In VitroGRO CP Medium (cat No Z99029) at 37° C. in anatmosphere with 5% CO₂ in a humidified incubator.

For transfection with siRNA, primary cynomolgus monkey cells were seededon Collagen coated plates (Fisher Scientific, cat. No. 08-774-5) at adensity of 3.5×10⁴ cells/well in 96-well plates and transfecteddirectly. Transfection of siRNA (eight 2-fold dilution series startingfrom 30 nM) in duplicates was carried out with lipofectamine 2000(Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) as describedby the manufacturer.

16 hours after transaction medium was changed to fresh In VitroGRO CPMedium with Torpedo Antibiotic Mix (In vitro Technologies, Inc, cat. NoZ99000) added.

24 hours alter medium change primary cynomolgus monkey cells were lysedand PCSK9 mRNA levels were quantified with the Quantigene Explore Kit(Genosprectra, Dumbarton Circle Fremont, USA, cat. No, QG-000-02)according to the protocol. PCSK9 mRNA levels were normalized to GAPDHmRNA. Normalized PCSK9/GAPDH ratios were then compared to PCSK9/GAPDHratio of lipofectamine 2000 only control.

Tables 1-2 (and FIG. 6) summarize the results and provides examples ofin vitro screens in different cell lines at different doses. Silencingof PCSK9 transcript was expressed as percentage of remaining transcriptat a given dose. Highly active sequences are those with less than 70%transcript remaining post treatment with a given siRNA at a dose lessthan or equal to 100 nm. Very active sequences are those that have lessthan 60% of transcript remaining after treatment with a dose less thanor equal to 100 nM. Active sequences are those that have less than 85%transcript remaining after treatment with a high dose (100 nM). Examplesof active siRNA's were also screened in vivo in mouse in lipidoidformulations as described below. Active sequences in vitro were alsogenerally active in vivo (See figure FIG. 6 example).

In vivo Efficacy Screen of PCSK9 siRNAs

Formulation Procedure

The lipidoid LNP-01 4HCl (MW 1487) (FIG. 1), Cholesterol(Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used toprepare lipid-siRNA nanoparticles. Stock solutions of each in ethanolwere prepared; LNP-01,133 mg/mL; Cholesterol, 25 mg/mL, PEG-CeramideC16,100 mg/mL. LNP-01, Cholesterol, and PEG-Ceramide C16 stock solutionswere then combined in a 42:48:10 molar ratio. Combined lipid solutionwas mixed rapidly with aqueous siRNA (in sodium acetate pH 5) such thatthe final ethanol concentration was 35-45% and the final sodium acetateconcentration was 100-300 mM. Lipid-siRNA nanoparticles formedspontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture was in some casesextruded through a polycarbonate membrane (100 nm cut-off) using athermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In othercases, the extrusion step was omitted. Ethanol removal and simultaneousbuffer exchange was accomplished by either dialysis or tangential flowfiltration. Buffer was exchanged to phosphate buffered saline (PBS) pH7.2.

Characterization of Formulations

Formulations prepared by either the standard or extrusion-free methodare characterized in a similar manner Formulations are firstcharacterized by visual inspection. They should be whitish translucentsolutions free from aggregates or sediment. Particle size and particlesize distribution of lipid-nanoparticles are measured by dynamic lightscattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particlesshould be 20-300 nm, and ideally; 40-100 nm in size. The particle sizedistribution should be unimodal. The total siRNA concentration in theformulation, as well as the entrapped fraction, is estimated using a dyeexclusion assay. A sample of the formulated siRNA is incubated with theRNA-binding dye Ribogreen (Molecular Probes) in the presence or absenceof a formulation disrupting surfactant, 0.5% Triton-X100. The totalsiRNA in the formulation is determined by the signal from the samplecontaining the surfactant, relative to a standard curve. The entrappedtraction is determined by subtracting the “free” siRNA content (asmeasured by the signal in the absence of surfactant) from the totalsiRNA content. Percent entrapped siRNA is typically >85%.

Bolus Dosing

Bolus dosing of formulated siRNAs in C57/BL6 mice (5/group, 8-10 weeksold, Charles River Laboratories, MA) was performed by tail veininjection using a 27G needle. SiRNAs were formulated in LNP-01 (and thendialyzed against PBS) at 0.5 mg/ml concentration allowing the deliveryof the 5 mg/kg dose in 10 μl/g body weight. Mice were kept under aninfrared lamp for approximately 3 min prior to dosing to ease injection.

48 hour post dosing mice were sacrificed by CO₂ asphyxiation. 0.2 mlblood was collected by retro-orbital bleeding and the liver washarvested and frozen in liquid nitrogen. Serum and livers were stored at−80° C.

Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder(SPEX CentriPrep, Inc) and powders stored at −80° C. until analysis.

PCSK9 mRNA levels were detected using the branched-DNA technology basedkit from QuantiGene Reagent System (Genospectra) according to theprotocol. 10-20 mg of frozen liver powders was lysed in 600 ul of 0.16ug/ml Proteinase K (Epicentre, #MPRK092) in Tissue and Cell LysisSolution (Epicentre, #MTC096H) at 65° C. for 3 hours. Then 10 ul of thelysates were added to 90 ul of Lysis Working Reagent (1 volume of stockLysis Mixture in two volumes of water) and incubated at 52° C. overnighton Genospectra capture plates with probe sets specific to mouse PCSK9and mouse GAPDH or cyclophilin B. Nucleic acid sequences for CaptureExtender (CE), Label Extender (LE) and blocking (BL) probes wereselected from the nucleic-acid sequences of PCSK9, GAPDH and cyclophilinB with the help of the QuantiGene ProbeDesigner Software 2.0(Genospectra, Fremont, Calif., USA, cat No. QG-Q002-G02). Chemoluminescence was read on a Victor2-Light (Perkin Elmer) as Relativelight units. The ratio of PCSK9 mRNA to GAPDH or cyclophilin B mRNA inliver lysates was averaged over each treatment group and compared to acontrol group treated with PBS or a control group treated with anunrelated siRNA (blood coagulation factor VII).

Total serum cholesterol in mouse serum was measured using the StanBioCholesterol LiquiColor kit (StanBio Laboratory, Boerne, Tex., USA)according to manufacturer's instructions. Measurements were taken on aVictor2 1420 Multilabel Counter (Perkin Elmer) at 495 nm.

EXAMPLES

32 PCSK9 siRNAs formulated in LNP-01 liposomes were tested in vivo in amouse model. The experiment was performed at 5 mg/kg siRNA dose and atleast 10 PCSK9 siRNAs showed more than 40% PCSK9 mRNA knock downcompared to a control group treated with PBS, while control grouptreated with an unrelated siRNA (blood coagulation factor VII) had noeffect (FIGS. 2-5). Silencing of PCSK9 transcript also coorelated with alowering of cholesterol in these animals (FIGS. 4-5). In addition therewas a strong coorelation between those molecules that were active invitro and those active in vivo (FIG. 6). Sequences containing differentchemical modifications were also screened in vitro (Tables 1 and 2) andin vivo. As an example, less modified sequences 9314 and 9318, and amore modified versions of that sequence 9314-(10792, 10793, and 10796);9318-(10794, 10795, 10797) were tested both in vitro (In primary monkeyhepatocytes) or in vivo (9314 and 10792) formulated in LNP-01. FIG. 7(also see Tables 1 and 2) shows that the parent molecules 9314 and 9318and the modified versions are all active in vitro. FIG. 8 as an exampleshows that both the parent 9314 and the more highly modified 10792sequences are active in vivo displaying 50-60% silencing of endogenousPCSK9 in mice. FIG. 9 further exemplifies that activity of otherchemically modified versions of the parents 9314 and 10792.

dsRNA Expression Vectors

In another aspect of the invention, PCSK9 specific dsRNA molecules thatmodulate PCSK9 gene expression activity are expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A. et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes canbe introduced as a linear construct a circular plasmid, or a viralvector, which can be incorporated and inherited as a transgeneintegrated into the host genome. The transgene can also be constructedto permit it to be inherited as an extrachromosomal plasmid (Gassmann,et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. In apreferred embodiment, a dsRNA is expressed as an inverted repeat joinedby a linker polynucleotide sequence such that the dsRNA has a stem andloop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others blown inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danes and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA.87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeuscechem, et al. 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application. WO 89/02468; PCT ApplicationWO 89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci, USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992. J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector of the invention may be a eukaryotic RNA polymerase I (e.g.liposomal RNA promoter), RNA polymerase II (e.g. CMV early promoter oractin promoter or U1 snRNA promoter) or generally RNA polymerase IIIpromoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter,for example the T7 promoter, provided the expression plasmid alsoencodes T7 RNA polymerase required for transcription from a T7 promoter.The promoter can also direct transgene expression, to the pancreas (see,e.g. the insulin regulatory sequence for pancreas (Bucchini et al.,1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical, inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single PCSK9 gene or multiple PCSK9 genes over a period of a weekor more are also contemplated by the invention. Successful introductionof the vectors of the invention into host cells can be monitored usingvarious known methods. For example, transient transfection. can besignaled with, a reporter, such as a fluorescent marker, such as GreenFluorescent Protein (GFP). Stable transfection. of ex vivo cells can beensured using markers that provide the transfected cell with resistanceto specific environmental factors (e.g., antibiotics and drugs), such ashygromycin B resistance.

The PCSK9 specific dsRNA molecules can also be inserted into vectors andused as gene therapy vectors for human patients. Gene therapy vectorscan be delivered to a subject by, for example, intravenous injection,local administration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

Those skilled in the art are familiar with methods and compositions inaddition to those specifically set out in the instant disclosure whichwill allow them to practice this invention to the full scope of theclaims hereinafter appended.

Mean percent re- maining mRNA trans- cript at siRNA concentration/inIC50 in position in cell type Cynomol- human 100 30 3 IC50 gous access.# SEQ SEQ nM/ nM/ nM/ 30 in monkey NM_17493 ID ID Duplex HepG HepG HepGnM/ HepG Hepatacyte 6 Sense strand sequence (5′-3′)¹ NO:Antisense-strand sequence (5′-3′)¹ NO: name 2 2 2 Hela 2 [nM] [nM]s 2-20 AGCGACGUCGAGGCGCUCATT 1 UGAGCGCCUCGACGCCGCUTT 2 AD- 35 15220 15-33CGCUCAUGGUUGCAGGCGGTT 3 CCGCCUGCAACCAUGAGCGTT 4 AD- 56 15275 16-34GCUCAUGGUUGCAGGCGGGTT 5 CCCGCCUGCAACCAUGAGCTT 6 AD- 70 15301 30-48GCGGGCGCCGCCGUUCAGUTT 7 ACUGAACGGCGGCGCCCGCTT 8 AD- 42 15276 31-49CGGGCGCCGCCGUUCAGUUTT 9 AACUGAACGGCGGCGCCCGTT 10 AD- 32 15302 32-50GGGCGCCGCCGUUCAGUUCTT 11 GAACUGAACGGCGGCGCCCTT 12 AD- 37 15303 40-58CCGUUCAGUUCAGGGUCUGTT 13 CAGACCCUGAACUGAACGGTT 14 AD- 30 15221 43-61UUCAGUUCAGGGUCUGAGCTT 15 GCUCAGACCCUGAACUGAATT 16 AD- 61 15413  82-100GUGAGACGGGCUCGGGCGGTT 17 CCGCCCGAGCCAGUCUCACTT 18 AD- 70 15304 100-118GGCCGGGACGCGUCGUUGCTT 19 GCAACGACGCGUCCCGGCCTT 20 AD- 36 15305 101-119GCCGGGACGCGUCGUUGCATT 21 UGCAACGACGCGUCCCGGCTT 22 AD- 20 15306 102-120CCGGGACGCGUCGUUGCAGTT 23 CUGCAACGACGCGUCCCGGTT 24 AD- 38 15307 105-123GGACGCGUCGUUGCAGCAGTT 25 CUGCUGCAACGACGCGUCCTT 26 AD- 50 135-153CCCCAGCCAGGAUUCCGCGTsT 27 CGCGGAAUCCUGGCUGGGATsT 28 AD- 74 89 9526135-153 ucctAGccAGGAuuccGcGTsT 29 CGCGGAAUCCUGGCUGGGATsT 30 AD- 97 9652136-154 CCCAGCCAGGAUUCCGCGCTsT 31 GCGCGGAAUCCUGGCUGGGTsT 32 AD- 78 9519136-154 cccAGccAGGAuuccGcGcTsT 33 GCGCGGAAUCCUGGCUGGGTsT 34 AD- 66 9646138-156 CAGCCAGGAUUCCGCGCGCTsT 35 GCGCGCGGAAGCCUGGCUGTsT 36 AD- 55 9523138-156 cAGccAGGAuuccGcGcGcTsT 37 GCGCGCGGAAUCCUGGCUGTsT 38 AD- 60 9649185-203 AGCUCCUGCACAGUCCUCCTsT 39 GGAGGACUGUGCAGGAGCUTsT 40 AD- 112 9569185-203 AGcuccaGcAcAGuccuccTsT 41 GGAGGACUGCGcAGGAGCUTsT 42 AD- 102 9695205-223 CACCGCAAGGCUCAAGGCGTT 43 CGCCUUGAGCCUUGCGGUGTT 44 AD- 75 15222208-226 CGCAAGGCUCAAGGCGCCGTT 45 CGGCGCCUUGAGCCUUGCGTT 46 AD- 78 15278210-228 CAAGGCUCAAGGCGCCGCCTT 47 GGCGGCGCCUUGAGCCUUGTT 48 AD- 83 15178232-250 GUGGACCGCGCACGGCCUCTT 49 GAGGCCGUGCGCGGUCCACTT 50 AD- 84 15308233-251 UGGACCGCGCACGGCCGCGTT 51 AGAGGCCGUGCGCGGCCCATT 52 AD- 67 15223234-252 GGACCGCGCACGGCCUCUATT 53 UAGAGGCCGUGCGCGGUCCTT 54 AD- 34 15309235-253 GACCGCGCACGGCCUCUAGTT 55 CUAGAGGCCGUGCGCGGUCTT 56 AD- 44 15279236-254 ACCGCGCACGGCCUCUAGGTT 57 CCUAGAGGCCGUGCGCGGUTT 58 AD- 63 15194237-255 CCGCGCACGGCCUCUAGGUTT 59 ACCUAGAGGCCGUGCGCGGTT 60 AD- 42 15310238-256 CGCGCACGGCCUCUAGGUCTT 61 GACCUAGAGGCCGUGCGCGTT 62 AD- 30 35311239-257 GCGCACGGCCUCUAGGUCUTT 63 AGACCUAGAGGCCGUGCGCTT 64 AD- 18 15392240-258 CGCACGGCCUCUAGGUCUCTT 65 GAGACCUAGAGGCCGUGCGTT 66 AD- 21 15312248-266 CUCUAGGUCUCCUCGCCAGTT 67 CUGGCGAGGAGACCUAGAGTT 68 AD- 19 15313249-267 UCUAGGUCUCCUCGCCAGGTT 69 CCUGGCGAGGAGACCUAGATT 70 AD- 81 15280250-268 CUAGGUCUCCUCGCCAGGATT 71 GCCUGGCGAGGAGACCUAGTT 72 AD- 82 15267252-270 AGGUCUCCUCGCCAGGACATT 73 GGUCCUGGCGAGGAGACCUTT 74 AD- 32 15314258-276 CCUCGCCAGGACAGCAACCTT 75 GGUUGCUGUCCUGGCGAGGTT 76 AD- 74 15315300-318 CGUCAGCUCCAGGCGGUCCTsT 77 GGACCGCCUGGAGCUGACGTsT 78 AD- 94 9624300-318 cGucAGcuccAGGcGGuccTsT 79 GGACCGCCUGGAGCUGACGTsT 80 AD- 96 9750301-319 GUCAGCUCCAGGCGGUCCUTsT 81 AGGACCGCCUGGAGCGGACTsT 82 AD- 43 669623 301-319 GucAGcuccAGGcGGuccuTsT 83 AGGACCGCCUGGAGCUGACTsT 84 AD- 1059749 370-388 GGCGCCCGUGCGCAGGAGGTT 85 CCUCCUGCGCACGGGCGCCTT 86 AD- 4815384 408-426 GGAGCUGGUGCUAGCCUUGTsT 87 CAAGGCUAGCACCAGCUCCTsT 88 AD- 3228 0.20 9607 408-426 GGAGcuGGuGcuAGccuuGTsT 89 cAAGGCuAGcACcAGCUCCTsT 90AD- 78 73 9733 411-429 GCUGGUGCUAGCCUUGCGUTsT 91 ACGCAAGGCUAGCACCAGCTsT92 AD- 23 28 0.07 9524 411-429 GcuGGuGcuAGccuuGcGuTsT 93ACGuAAGGCuAGcACcAGCTsT 94 AD- 91 90 9650 412-430 CUGGUGCUAGCCUUGCGUUTsT95 AACGCAAGGCUAGCACCAGTsT 96 AD- 23 32 9520 412-430CUGGUGCUAGCCUUGCGUUTsT 97 AACGCAAGGCUAGCACCAGTsT 98 AD- 23 9520 412-430cuGGuGcuAGccuuGcGucTsT 99 AACGcAAGGCuAGcACcAGTsT 100 AD- 97 108 9646416-434 UGCCAGCCUUGCGUUCCGATsT 101 UCGGAACGCAAGGCUAGCATsT 102 AD- 379608 416-434 uGcuAGccuuGcGuuccGATsT 103 UCGGAACGcAAGGCuAGcATsT 104 AD-91 9734 419-437 UAGCCUUGCGUUCCGAGGATsT 105 UCCUCGGAACGCAAGGCUATsT 106AD- 32 9546 419-437 uAGccuuGcGuuccGAGGATsT 107 UCCUCGGAACGcAAGGCuATsT108 AD- 57 9672 439-457 GACGGCCUGGCCGAAGCACTT 109 GUGCUUCGGCCAGGCCGUCTT110 AD- 54 15385 447-465 GGCCGAAGCACCCGAGCACTT 111 GUGCUCGGGUGCUUCGGCCTT112 AD- 31 15393 448-466 GCCGAAGCACCCGAGCACGTT 113 CGUGCUUGGGUGCUUCGGCTT114 AD- 37 15316 449-467 CCGAAGCACCCGAGCACCGTT 115 CCGUGCUCGGGUGCUUCGGTT116 AD- 37 15317 458-476 CCGAGCACGGAACCACAGCTT 117 GCUGUGGUUCCGUGCUCGGTT118 AD- 63 484-502 CACCGCUGCGCCAAGGAUCTT 119 GAUCCUUGGCGCAGCGGUGTT 120AD- 45 15195 486-504 CCGCUGCGCCAAGGAUCCGTT 121 CGGAUCCUUGGCGCAGCGGTT 122AD- 57 15224 487-505 CGCUGCGCCAAGGAUCCGUTT 123 ACGGAUCCUUGGCGCAGCGTT 124AD- 42 15188 489-507 CUGCGCCAAGGAUCCGUGGTT 125 CCACGGAUCCUUGGCGCAGTT 126AD- 51 15225 500-518 AUCCGUGGAGGUUGCCUGGTT 127 CCAGGCAACCUCCACGGAUTT 128AD- 89 15281 509-527 GGUUGCCUGGCACCUACGUTT 129 ACGUAGGUGCCAGGCAACCTT 130AD- 75 15282 542-560 AGGAGACCCACCUCUCGCATT 131 UGCGAGAGGUGGGUCUCCUTT 132AD- 61 15319 543-561 GGAGACCCACCUCUGGCAGTT 133 CUGCGAGAGGUGGGUCUCCTT 134AD- 56 15226 544-562 GAGACCCACCUCUCGCAGUTT 135 ACUGCGAGAGGUGGGUCUCTT 136AD- 25 15271 549-567 CCACCUCUCGCAGUCAGAGTT 137 CUCUGACUGCGAGAGGUGGTT 138AD- 25 15283 552-570 CCUCUCGCAGUCAGAGCGCTT 139 GCGCUCUGACUGCGAGAGGTT 140AD- 64 15284 553-571 CUCUCGCAGUCAGAGCGCATT 141 UGCGCUCUGACUGCGAGAGTT 142AD- 37 15189 554-572 UCUCGCAGUCAGAGCGCACTT 143 GUGCGCUCUGACUGCGAGATT 144AD- 62 15227 555-573 CUCGCAGUCAGAGCGCACUTsT 145 AGUGCGCUCUGACUGCGAGTsT146 AD- 31 29 0.20 9547 555-573 cucGcAGucAGAGcGcAcuTsT 147AGUGCGCUCUGACUGCGAGTsT 148 AD- 56 57 9673 558-576 GCAGUCAGAGCGCACUGCCTsT149 GGCAGUGCGCUCUGACUGCTsT 150 AD- 54 60 9548 558-576GcAGucAGAGcGcAcuGccTsT 151 GGcAGUGCGCUCUGACUGCTsT 152 AD- 36 57 9674606-624 GGGAUACCUCACCAAGAUCTsT 153 GAUCUUGGUGAGGUAUCCCTsT 154 AD- 609529 606-624 GGGAuAccucAccAAGAucTsT 155 GAUCUUGGUGAGGuAUCCCTsT 156 AD-140 9655 659-677 UGGUGAAGAUGAGUGGGGATsT 157 UCGCCACUCAUCUUCACCATsT 158AD- 27 31 0.27 9605 659-677 uGGuGAAGAuGAGuGGcGATsT 159UCGCcACUcAUCCUUACcATsT 160 AD- 31 31 0.32 9731 663-681GAAGAUGAGUGGCGACCUGTsT 161 CAGGUCGCCACUCAUCUUCTsT 162 AD- 37 9596663-681 GAAGAuGAGuGGcGAccuGTsT 163 cAGGUCGCcACUcAUCUUCTsT 164 AD- 769722 704-722 CCCAUGUCGACUACAUCGATsT 165 UCGAUGUAGUCGACAUGGGTsT 166 AD-42 9583 704-722 cccAuGucGAcuAcAucGATsT 167 UCGAUGuAGUCGAcAUGGGTsT 168AD- 104 9709 718-736 AUCGAGGAGGACUCCUCUGTsT 169 CAGAGGAGUCCUCCUCGAUTsT170 AD- 113 9579 718-736 AucGAGGAGGAcuccucuGTsT 171cAGAGGAGUCCUCCUCGAUTsT 172 AD- 81 9705 758-776 GGAACCUGGAGCGGAUUACTT 173GUAAUCCGCUCCAGGUUCCTT 174 AD- 32 15394 759-777 GAACCUGGAGCGGAUUACCTT 175GGUAAUCCGCUCCAGGUUCTT 176 AD- 72 15196 760-778 AACCUGGAGCGGAUUACCCTT 177GGGUAAUCCGCUCCAGGUUTT 178 AD- 85 15197 777-795 CCCUCCACGGUACCGGGCGTT 179CGCCCGGUACCGUGGAGGGTT 180 AD- 71 15198 782-800 CACGGUACCGGGCGGAUGATsT181 UCAUCCGCCCGGUACCGUGTsT 182 AD- 66 71 9609 782-800cAcGGuAccGGGcGGAuGATsT 183 UcAUCCGCCCGGuACCGUGTsT 184 AD- 115 9735783-801 ACGGUACCGGGCGGAUGAATsT 185 UUCAUCCGCCCGGUACCGUTsT 186 AD- 1459537 783-801 AcGGuAccGGGcGGAuGAATsT 187 UUcAUCCGCCCGGuACCGUTsT 188 AD-102 9663 784-802 CGGUACCGGGCGGAUGAAUTsT 189 AUUCAUCCGCCCGGUACCGTsT 190AD- 113 9528 784-802 cGGuAccGGGcGGAuGAAuTsT 191 AUUcAUCCGCCCGGuACCGTsT192 AD- 107 9654 785-803 GGUACCGGGCGGAUGAAUATsT 193UAUUCAUCCGCCCGGUACCTsT 194 AD- 49 9615 785-803 GGuAccGGGcGGAuGAAcATsT195 uAUUcAUCCGCCCGGuACCTsT 196 AD- 92 9641 786-804GUACCGGGCGGAUGAAUACTsT 197 GUAUUCACCCGCCCGGUACTsT 198 AD- 57 9514786-804 GuAccGGGcGGAuGAAuACTsT 199 GuAUUcAUCCGCCCGGuACTsT 200 AD- 899640 788-806 ACCGGGCGGAUGAAUACCATsT 201 UGGUAUUCAUCCGCCCGGUTsT 202 AD-75 9530 788-806 AccGGGcGGAuGAAuAccATsT 203 UGGuAUUcAUCCGCCCGGUTsT 204AD- 77 9656 789-807 CCGGGCGGAUGAAUACCAGTsT 205 CUGGUAUCCAUCCGCCCGGTsT206 AD- 79 80 9538 789-807 ccGGGcGGAuGAAuAccAGTsT 207CUGGuAUUcAUCCGCCCGGTsT 208 AD- 53 9664 825-843 CCUGGUGGAGGUGUAUCUCTsT209 GAGAUACACCUCCACCAGGTsT 210 AD- 69 83 9598 825-843ccuGGuGGAGGuGuAucucTsT 211 GAGAuAcACCUCcACcAGGTsT 212 AD- 127 9724826-844 CUGGUGGAGGUGUAUCUCCTsT 213 GGAGAUACACCUCCACCAGTsT 214 AD- 58 889625 826-844 cuGGuGGAGGuGuAucuccTsT 215 GGAGAuAcACCUGcACcAGTsT 216 AD-60 9751 827-845 UGGUGGAGGUGUAUCUCCUTsT 217 AGGAGAUACACCUCCACCATsT 218AD- 46 9556 827-845 uGGuGGAGGuGuAucuccuTsT 219 AGGAGAuAcACCUGcACcATsT220 AD- 38 9682 828-846 GGUGGAGGUGUAUCUCCUATsT 221UAGGAGAUACACCUCCACCTsT 222 AD- 56 63 9539 828-846 GGuGGAGGuGuAucuccuATsT223 uAGGAGAuAcACCUGcACCTsT 224 AD- 83 9665 831-849GGAGGUGUAUCUCCUAGACTsT 225 GUCUAGGAGAUACACCUCCTsT 226 AD- 36 9517831-849 GGAGGcGuAucuccuAGAcTsT 227 GUCuAGGAGAuAcACCUCCTsT 228 AD- 409643 833-851 AGGUGUAUCUCCUAGACACTsT 229 GUGUCUAGGAGAUACACCUTsT 230 AD-36 34 0.04 9610 833-851 AGGuGuAucuccuAGAcAcTsT 231GUGUCuAGGAGAuAcACCUTsT 232 AD- 22 29 0.04 9736 833-851AfgGfuGfUAfuCfuCfcUfaGfaCfaCTTsT 233 p-gUfgUfcUfaGfgAfgAfaAfcAcCfaTsT234 AD- 33 14681 833-851 AGGUfGUfAUfCfUfCfCfUfAGACfACfTsT 235GGfGUfCfUfAGGAGAUfACfACfCfUfTsT 236 AD- 27 14691 833-851AgGuGuAaCaCcUaGuCaCTsT 237 p-gUfgUfcUfaGfgAfgAfuAfcAfcCfuTsT 238 AD- 3214701 833-851 AgGuGuAaCuCcUaGaCaCTsT 239 GUfGUfCfUfAGGAGAUfACfACfCfUfTsT240 AD- 33 14711 833-851 AfgGfuGfuAfuCfuCfcUfaGfaCfaCfTsT 241GUGUCuaGGagAUACAccuTsT 242 AD- 22 14721 833-851AGGUfGUfAUfCfUfCfCfUfAGACfACfTsT 243 GUGUCuaGGagAUACAccuTsT 244 AD- 2114731 833-851 AgGuGuAuCuCcUaGaCaCTsT 245 GUGUCuaGGagAUACAccuTsT 246 AD-23 14741 833-851 GfcAfcCfcUfcAfuAfgGfcCfuGfgAfTsT 247p-uCfcAfgGgcCfuAfcGfaGfgCfuGfcTsT 248 AD- 37 15087 833-851GCfACfCfCfUfCfAUfAGGCfCfUfGGATsT 249 UfCfCfAGGCfCfUfAUfGAGGGUfGCfTsT 250AD- 51 15097 833-851 GcAcCcUcAuAgGcCuGgATsT 251p-aCfcAfgGfcCfuAfaGfaGfgGfaGfcTsT 252 AD- 26 15107 833-851GcAcCcUcAuAgGcCuGgATsT 253 UfCfCfAGGCfCfUfAUfGAGGGUfGCfTsT 254 AD- 2815117 833-851 GfcAfcCfcUfcAfuAfgGfcCfuGfgAfTsT 255UCCAGgcCUauGAGGGugcTsT 256 AD- 33 15127 833-851GCfACfCfCfuFCfAUfAGGCfCfUfGGATsT 257 UCCAGgcCUauGAGGGugcTsT 258 AD- 5415137 833-851 GcAcCcUcAuAgGcCuGgATsT 259 UCCAGgcCUauGAGGGugcTsT 260 AD-52 15147 836-854 UGUAUCUCCUAGACACCAGTsT 261 CUGGUGUCUAGGAGAUACATsT 262AD- 94 9516 836-854 uGuAucuccuAGAcAccAGTsT 263 CUGGUGUCuAGGAGAuAcATsT264 AD- 105 9642 840-858 UCUCCUAGACACCAGCAUATsT 265UAUGCUGGUGUCUAGGAGATsT 266 AD- 46 51 9562 840-858 ucuccuAGAcAccAGcAuATsT267 uAUGCUGGUGGCuAGGAGATsT 268 AD- 26 34 4.20 9688 840-858UfcUfcCfuAfgAfcAfcCfaGfcAfuAfTsT 269 p-uAfuGfcUfgGfuGfuCfuAfgGfaGfaTsT270 AD- 38 14677 840-858 UfCfCfCfCfUfAGACfACfCfAGCfAUfATsT 271UfAUfGCfUfGGUfGUfCfUfAGGAGATsT 272 AD- 52 14687 840-858UcUcCuAgAcAcCaGcAuATsT 273 p-uAfuGfcUfgGfuGfuCfuAfgGfaGfaTsT 274 AD- 3514697 840-858 UcUcCuAgAcAcCuGcAuATsT 275 UfAUfGCfUfGGUfGUfCfUfAGGAGATsT276 AD- 58 14707 840-858 UfcUfcCfuAafAfcAfcCfaGfcAfuAfTsT 277UAUGCugGUguCUAGGagaTsT 278 AD- 42 14717 840-858UfCfCfCfCfUfAGACfACfCfAGCfAUfATsT 279 UAUGCugGUguCUAGGugaTsT 280 AD- 5014727 840-858 UcGcCuAgAcAcCaGcAcATsT 281 UAGCGugGUguCUAGGagaTsT 282 AD-32 14737 840-858 AfgGfcCfuGfgAfgUfuUfaUfuCfgGfTsT 283p-cCfgAfaUfaAfaCfaCfcAfgGfcCfuTsT 284 AD- 16 15083 840-858AGGCfCfUfGGAGUfUfCfAUfUfCfGGTsT 285 CfCfGAAUfAAACfGfCfCfAGGCfCfUfTsT 286AD- 24 15093 840-858 AgGcCuGgAgUuUaUuCgGTsT 287p-cCfgAfaUfaAfuCfuCfcAfgGfcCfuTsT 288 AD- 11 15103 840-858AfgGfcCfuGfgAfgUfuUfaUfuCfgGfTsT 289 CfCfGAAUfAAACfUfCfCfAGGCfCfUfTsT290 AD- 34 15113 840-858 AfgGgcCfuGfgAfgUfuUfaUfuCfgGfTsT 291CCGAAuaAAcuCCAGGccuTsT 292 AD- 19 15123 840-858AGGCfCfUfGGAGUfUfUfAUfUfCfGGTsT 293 CCGAAuaAAcuCCAGGccuTsT 294 AD- 1515133 840-858 AgGcCuGgAgUcUaUcCgGTsT 295 CCGAAuaAAcuCCAGGccuTsT 296 AD-16 15143 841-859 CUCCUAGACACCAGCAUACTsT 297 GUAUGCUGGUGUCUAGGAGTsT 298AD- 50 9521 841-859 cuccuAGAcAccAGcAuAcTsT 299 GuAUGCUGGUGUCuAGGAGTsT300 AD- 62 9647 842-860 UCCUAGACACCAGCAUACATsT 301UGUAUGCUGGUGUCUAGGATsT 302 AD- 48 9611 842-860 uccuAGAcAccAGcAuAcATsT303 UGcAUGCUGGUGUCuAGGATsT 304 AD- 68 9737 843-861CCUAGACACCAGCAUACAGTsT 305 CUGUAUGCUGGUGUCUAGGTsT 306 AD- 46 55 9592843-861 ccUAGAcAccAGcAuAcAGTsT 307 CUGuAUGCUGGUGUCuAGGTsT 308 AD- 789718 847-865 GACACCAGCAUACAGAGUGTsT 309 CACUCUGUAUGCUGGUGUCTsT 310 AD-64 9561 847-865 GAcAccAGcAuAcAGAGuGTsT 311 cACGCUGuAUGCUGGUGUCTsT 312AD- 84 9687 855-873 CAUACAGAGUGACCACCGGTsT 313 CCGGUGGUCACUCUGUACGTsT314 AD- 42 41 2.10 9762 855-873 cAuAcAGAGuGAccAccGGTsT 315CCGGUGGUcACUCUGuAUGTsT 316 AD- 9 28 0.40 9762 860-878AGAGUGACCACCGGGAAAUTsT 317 AUUUCCCGGUGGUCACUCUTsT 318 AD- 45 9540860-878 AGAGuGAccAccGGGAAAUTsT 319 AUUUCCCGGUGGUcACUCUTsT 320 AD- 819666 861-879 GAGUGACCACCGGGAAAUCTsT 321 GAUUUCCCGGUGGUCACUCTsT 322 AD-48 73 9535 861-879 GAGuGAccAccGGGAAAccTsT 323 GAUUUCCCGGUGGUcACUCTsT 324AD- 83 9661 863-881 GUGACCACCGGGAAAUCGATsT 325 UCGAUUUCCCGGUGGUCACTsT326 AD- 35 9559 863-881 GuGAccAccGGGAAAucGATsT 327UCGAUUUCCCGGUGGUcACTsT 328 AD- 77 9685 865-883 GACCACCGGGAAAUCGAGGTsT329 CCUCGAUUUCCCGGUGGUCTsT 330 AD- 100 9533 866-884ACCACCGGGAAAUCGAGGGTsT 331 CCUCGAUUUCCCGGUGGUCTsT 332 AD- 88 9659866-884 ACCACCGGGAAAUCGAGGGTsT 333 CCCUCGAUUUCCCGGUGGUTsT 334 AD- 1229612 866-884 AccAccGGGAAAucGAGGGTsT 335 CCCUCGAUUUCCCGGUGGUTsT 336 AD-83 9738 867-885 CCACCGGGAAAUCGAGGGCTsT 337 GCCCUCGAUUUCCCGGUGGTsT 338AD- 75 96 9557 867-885 cCAccGGGAAAucGAGGGcTsT 339 GCCCUCGAUUUCCCGGUGGTsT340 AD- 48 9683 875-893 AAAUCGAGGGCAGGGUCAUTsT 341AUGACCCUGCCCUCGAUUUTsT 342 AD- 31 32 0.53 9531 875-893AAAucGAGGGcAGGGucAuTsT 343 AUGACCCUGCCCUCGAUUUTsT 344 AD- 23 29 0.669657 875-893 AfaAfuCfgAfgGfgCfaGfggfcCfaUfTsT 345p-aUfgAfcCfcUfgCfcCfuCfgAfuUfuTsT 346 AD- 81 14673 875-893AAAUfCfGAGGGCfAGGGUfCfAUfTsT 347 AUfGACfCfCfUfGCfCfCfUfCfGAUfUfTs 348AD- 56 T 14683 875-893 AaAuCgAgGgCaGgGuCaUTsT 349p-aUfgAfcCfcUfgCfcCfuCfGAfuUfuTsT 350 AD- 56 14693 875-893AaAuCgAgGgCaGgGuCaUTsT 351 AUfGACfCfCfUfGCfCfCfUfGAUfUfUfTs 352 AD- 68 T14703 875-893 AfaAfuCfGAfgGfgCfaGfgGfcCfaUfTsT 353AUGACccUCccCUCGAuuuTsT 354 AD- 55 14713 875-893AAAUfCfGAGGGCfAGGGUfCfAUfTsT 355 AUGACccUGccCUCGAuuuTsT 356 AD- 24 1472387-893 AuAuCgAggGcaGgGuCaUTsT 357 AUGACccUGccCUCGAuuuTsT 358 AD- 3414733 85-893 CfgGgcAfcCfcUfCAfuAfGGfCCfuGfTsT 359p-cAfgGgcCfuAfuGfaGfgGfuGfcCfgTsT 360 AD- 85 15079 875-893CfGGCfACfCfCfUfCfAUfAGGCfCfUfGTsT 361 CfAGGCfCfUfAUfGAGGGfGCfCfGTsT 362AD- 54 15089 875-893 CgGcAcCcUcAuAgGcCuGTsT 363p-cAfgGfcCfuAfuGfaGfgGfuGfcCfgTsT 364 AD- 70 15099 875-893GcGcAcCcUcAuAgGcCcGTsT 365 CfAGGCfCfUfAUfGAGGGUfGCfCfGTsT 366 AD- 6715109 875-893 CfgGgcAfcCfcUfcAfuAfgGgcCfuGfTsT 367CAGGCcuAUgaGGGUGccgTsT 368 AD- 67 15119 875-893CfGGCfACfCfCfUfCfAUfAGGCfCfUfGTsT 369 CAGGCcuAUgaGGGUGccgTsT 370 AD- 5715129 875-893 CgGcAcCcUcAuAgGcCuGTsT 371 CAGGCcuAGgaGGGUGccgTsT 372 AD-69 15139 877-895 AUCGAGGGCAGGGUCAUGGTsT 373 CCAUGACCCUGCCCUCGAUTsT 374AD- 160 9542 877-895 AucGAGGGcAGGGucAuGGTsT 375 CcAUGACCCUGCCCUCGAUTsT376 AD- 92 9668 878-896 cGAGGGcAGGGucAuGGucTsT 377GACcAUGACCCUGCCCUCGTsT 378 AD- 109 9739 880-898 GAGGGCAGGGUCAUGGUCATsT379 UGACCAUGACCCUGCCCUCTsT 380 AD- 56 83 9637 880-898GAGGGcAGGGucAuGGucATsT 381 UGACcAUGACCCUGCCCUCTsT 382 AD- 79 9763882-900 GGGCAGGGUCAUGGUCACCTsT 383 GGUGACCAUGACCCUGCCCTsT 384 AD- 829630 882-900 GGGcAGGGucAuGGucAccTsT 385 GGUGACcAUGACCCUGCCCTsT 386 AD-63 9756 885-903 CAGGGUCAUGGUCACCGACTsT 387 GUCGGUGACCAUGACCCUGTsT 388AD- 55 9593 885-903 cAGGGucAcGGucAccGAcTsT 389 GUCGGUGACcAUGACCCUGTsT390 AD- 115 9719 886-904 AGGGUCAUGGUCACCGACUTsT 391AGUCGGUGACCAUGACCCUTsT 392 AD- 111 9601 886-904 AGGGucAuGGucAccGAcuTsT393 AGUCGGUGACcAUGACCCUTsT 394 AD- 118 9727 892-910AUGGUCACCGACUUCGAGATsT 395 UCUCGAAGUCGGUGACCAUTsT 396 AD- 36 42 1.609573 892-910 AuGGucAccGAcuucGAGATsT 397 UCUCGAAGUCGGUGACcAUTsT 398 AD-32 36 2.50 9699 899-917 CCGACUUCGAGAAUGUGCCTT 399 GGCACAUUCUCGAAGUCGGTT400 AD- 26 15228 921-939 GGAGGACGGGACCCGCUUCTT 401 GAAGCGGGUCCCGUCCUCCTT402 AD- 53 15395  993-1011 CAGCGGCCGGGAUGCCGGTsT 403GCCGGCAUCCCGGCCGCUGTsT 404 AD- 126 9602 993-1011 cAGcGGccGGGAuGccGGcTsT405 GCCGGcAUCCCGGCCGCUGTsT 406 AD- 94 9728 1020-1038GGGUGCCAGCAUGCGCAGCTT 407 GCUGCGCAUGCUGGCACCCTT 408 AD- 45 153861038-1056 CCUGCGCGUGCUCAACUGCTsT 409 GCAGUUGAGCACGCGCAGGTsT 410 AD- 1129580 1038-1058 UGCGCGUGCUCAACUGCCATsT 411 GcAGUUGAGcACGCGcAGGTsT 412 AD-86 9706 1040-1058 UGCGCGUGCUCAACUGCCATsT 413 UGGCAGUUGAGCACGCGCATsT 414AD- 35 9581 1040-1058 uGcGcGuGcucAAcuGccATsT 415 UGGcAGUUGAGcACGCGcATsT416 AD- 81 9707 1042-1060 CGCGUGCUCAACUGCCAAGTsT 417CUUGGCAGUUGAGCACGCGTsT 418 AD- 51 9543 1042-1060 cGcGuGcucAAcuGccAAGTsT419 CUUGGcAGUUGAGcACGCGTsT 420 AD- 97 9669 1053-1071CUGCCAAGGGAAGGGCACGTsT 421 CGUGCCCUUCCCUUGGCAGTsT 422 AD- 74 95741053-1071 cuGccAAGGGAAGGGcAcGTsT 423 CGUGCCCUUCCCUUGGcAGTsT 424 AD- 97001057-1075 CAAGGGAAGGGCACGGUUATT 425 UAACCGUGCCCUUCCCUUGTT 426 AD- 2615320 1058-1076 AAGGGAAGGGCACGGUUAGTT 427 CUAACCGUGCCCUUCCCUUTT 428 AD-34 15321 1059-1077 AGGGAAGGGCACGGUUAGCTT 429 GCUAACCGUGCCCUUCCCUTT 430AD- 64 15199 1060-1078 GGGAAGGGCACGGUUAGCGTT 431 CGCUAACCGUGCCCUUCCCTT432 AD- 86 15167 1061-1079 GGAAGGGCACGGUUAGCGGTT 433CCGCUAACCGUGCCCUUCCTT 434 AD- 41 15164 1062-1080 GAAGGGCACGGUUAGCGGCTT435 GCCGCUAACCGUGCCCUUCTT 436 AD- 43 15166 1063-1081AAGGGCACGGUUAGCGGCATT 437 UGCCGCUAACCGUGCCCUUTT 438 AD- 64 153221064-1082 AGGGCACGGUUAGCGGCACTT 439 GCGCCGCUAACCGUGCCCUTT 440 AD- 4615200 1068-1086 CACGGUUAGCGGCACCCUCTT 441 GAGGGUGCCGCUAACCGUGTT 442 AD-27 15213 1069-1087 ACGGUUAGCGGCACCCUCATT 443 UGAGGGUGCCGCUAACCGUTT 444AD- 44 15229 1072-1090 GUUAGCGGCACCCUCAUAGTT 445 CUAUGAGGGUGCCGCUAACTT446 AD- 49 15215 1073-1091 UUAGCGGCACCCUCAUAGGTT 447CCUAUGAGGGUGCCGCUAATT 448 AD- 101 15214 1076-1094 GCGGCACCCUCAUAGGCCUTsT449 AGGCCUAUGAGGGUGCCGCTsT 450 AD- 15 32 0.98 9315 1079-1097GCACCCUCAUAGGCCUGGATsT 451 UCCAGGCCUAUGAGGGUGCTsT 452 AD- 35 51 93261085-1103 UCAUAGGCCUGGAGUUUAUTsT 453 AUAAACUCCAGGCCUAUGATsT 454 AD- 1437 0.40 9318 1090-1108 GGCCUGGAGUUUAUUCGGATsT 455 UCCGAAUAAACUCCAGGCCTsT456 AD- 14 33 9323 1091-1109 GCCUGGAGUUUAUUCGGAATsT 457UUCCGAAUAAACUCCAGGCTsT 458 AD- 11 22 0.04 9314 1091-1109GccuGGAGuuuAuccGGAATsT 459 UUCCGAAuAAACUCcAGGCTsT 460 AD- 0.10 0.3010792 1091-1109 GccuGGAGuuuAuucGGAATsT 461 UUCCGAAUAACUCCAGGCTsT 462 AD-0.1 0.1 10796 1093-1111 CUGGAGUUUAUUCGGAAAATsT 463UUUUCCGAAUAAACUCCAGTsT 464 AD- 101 9638 1093-1111 cuGGAGuuuAuucGGAAAATsT465 UUUUCCGAAuAACUCcAGTsT 466 AD- 112 9764 1095-1113GGAGUUUAUUCGGAAAAGCTsT 467 GCUUUUCCGAAUAAACUCCTsT 468 AD- 53 95251095-1113 GGAGuuuAuucGGAAAAGcTsT 469 GCUUUUCCGAAuAAACUCCTsT 470 AD- 589651 1096-1114 GAGUUUAUUCGGAAAAGCCTsT 471 GGCUUUUCCGAAUAAACUCTsT 472 AD-97 9560 1096-1114 GACuuuAuccGGAAAAGcCTsT 473 GGCUUUUCCGAAuAAACUCTsT 474AD- 111 9686 1100-1118 UUAUUCGGAAAAGCCAGCUTsT 475 AGCUGGCUUUUCCGAAUAATsT476 AD- 157 9536 1100-1118 uuAuucGGAAAAGccAGcuTsT 477AGCUGGCUUUUCCGAAuAATsT 478 AD- 81 9662 1154-1172 CCCUGGCGGGUGGGUACAGTsT479 CUGUACCCACCCGCCAGGGTsT 480 AD- 52 68 9584 1154-1172cccuGGcGGGuGGGuAcAGTsT 481 CUGcACCcACCCGCcAGGGTsT 482 AD- 111 97101155-1173 CCUGGCGGGUGGGUACAGCTT 483 GCUGUACCCACCCGCCAGGTT 484 AD- 6215323 1157-1175 UGGCGGGUGGGUACAGCCGTsT 485 CGGCUGUACCCACCCGCCATsT 486AD- 91 9551 1157-1175 uGGcGGGuGGGuAcAGccGTsT 487 CGGCUGuACCcACCCGCcATsT488 AD- 62 9677 1158-1176 GGCGGGUGGGUACAGCCGCTT 489GCGGCUGUACCCACCCGCCTT 490 AD- 52 15230 1162-1180 GGUGGGUACAGCCGCGUCCTT491 GGACGCGGCUGUACCCACCTT 492 AD- 25 15231 1164-1182UGGGUACAGCCGCGUCCUCTT 493 GAGGACGCGGCUGUACCCATT 494 AD- 36 152851172-1190 GCCGCGUCCUCAACGCCGCTT 495 GCGGCGUUGAGGACGCGGCTT 496 AD- 2715396 1173-1191 CCGCGUCCUCAACGCCGCCTT 497 GGCGGCGUUGAGGACGCGGTT 498 AD-36 15397 1216-1234 GUCGUGCUGGUCACCGCUGTsT 499 CAGCGGUGACCAGCACGACTsT 500AD- 112 9600 1216-1234 GucGuGcuGGucAccGcuGTsT 501 cAGCGGUGACcAGcACGACTsT502 AD- 95 9726 1217-1235 UCGUGCUGGUCACCGCUGCTsT 503GCAGCGGUGACCAGCACGATsT 504 AD- 107 9606 1217-1235 ucGuGcuGGucAccGcuGcTsT505 GcAGCGGUGACcAGcACGATsT 506 AD- 105 9732 1223-1241UGGUCACCGCUGCCGGCAATsT 507 UUGCCGGCAGCGGUGACCATsT 508 AD- 56 75 96331223-1241 uGGucAccGcuGccGGcAATsT 509 UUGCCGGcAGCGGUGACcATsT 510 AD- 1119759 1224-1242 GGUCACCGCUGCCGGCAACTsT 511 GUUGCCGGCAGCGGUGACCTsT 512 AD-66 9588 1224-1242 GGucAccGcuGccGGcAAcTsT 513 GUUGCCGGcAGCGGUGACCTsT 514AD- 106 9714 1227-1245 CACCGCUGCCGGCAACUUCTsT 515 GAAGUUGCCGGCAGCGGUGTsT516 AD- 67 85 9589 1227-1245 cAccGuuGccGGcAAcuucTsT 517GAAGUUGCCGGcAGCGGUGTsT 518 AD- 113 9715 1229-1247 CCGCUGCCGGCAACUUCCGTsT519 CGGAAGUUGCCGGCAGCGGTsT 520 AD- 120 9575 1229-1247cCGcuGccGGcAAcuuccGTsT 521 CGGAAGUUGCCGGcAGCGGTsT 522 AD- 100 97011230-1248 CGCUGCCGGCAACUUCCGGTsT 523 CCGGAAGUUGCCGGCAGCGTsT 524 AD- 1039563 1230-1248 cGcuGccGGcAAuuuccGGTsT 525 CCGGAAGUUGCCGGcAGCGTsT 526 AD-81 9689 1231-1249 GCUGCCGGCAACUUCCGGGTsT 527 CCCGGAAGUUGCCGGCAGCTsT 528AD- 80 95 9594 1231-1249 GcuGccGGcAAcuuccGGGTsT 529CCCGGAAGUGGCCGGcAGCTsT 530 AD- 92 9720 1236-1254 CGGCAACUUCCGGGACGAUTsT531 AUCGUCCCGGAAGUUGCCGTsT 532 AD- 83 9585 1236-1254cGGcAAcuuccGGGAcGAuTsT 533 AUCGUCCCGGAAGUUGCCGTsT 534 AD- 122 97111237-1255 GGCAACUUCCGGGACGAUGTsT 535 CAUCGUCCCGGAAGUUGCCTsT 536 AD- 1009614 1237-1255 GGcAAcuuccGGGAcGAuGTsT 537 cAUCGUCCCGGAAGUUGCCTsT 538 AD-198 9740 1243-1261 UUCCGGGACGAUGCCUGCCTsT 539 GGCAGGCAUGGUCCCGGAATsT 540AD- 116 9615 1243-1261 uuccGGGAcGAuGccuGcCTsT 541 GGcAGGcAUCGUCCCGGAATsT542 AD- 130 9741 1248-1266 GGACGAUGCCUGCCUCUACTsT 543GUAGAGGCAGGCAUCGUCCTsT 544 AD- 32 30 9534 1248-1266GGACGAUGCCUGCCUCUACTsT 545 GUAGAGGCAGGCAUCGUCCTsT 546 AD- 32 95341248-1266 GGAcGAuGccuGccucuAcTsT 547 GuAGAGGcAGGcAUCGUCCTsT 548 AD- 8979 9660 1279-1297 GCUCCCGAGGUCAUCACAGTT 549 CUGUGAUGACCUCGGGAGCTT 550AD- 46 15324 1280-1298 CUCCCGAGGUCAUCACAGUTT 551 ACUGUGAUGACCUCGGGAGTT552 AD- 19 15232 1281-1299 UCCCGAGGUCAUCACAGUUTT 553AACUGUGAUGACCUCGGGATT 554 AD- 25 15233 1314-1332 CCAAGACCAGCCGGUGACCTT555 GGUCACCGGCUGGUCUUGGTT 556 AD- 59 15234 1315-1333CAAGACCAGCCGGUGACCCTT 557 GGGUCACCGGCUGGUCUUGTT 558 AD- 109 152861348-1366 ACCAACUUUGGCCGCUGUGTsT 559 CACAGCGGCCAAAGUUGGUTsT 560 AD- 1229590 1348-1366 AccAAcuuuGGccGcuGuGTsT 561 cAcAGCGGCcAAAGUUGGUTsT 562 AD-114 9716 1350-1368 CAACUUUGGCCGCUGUGUGTsT 563 CACACAGCGGCCAAAGUUGTsT 564AD- 34 9632 1350-1368 cAAcuuuGGccGcuGuGuGTsT 565 cAcAcAGCGGCcAAAGUUGTsT566 AD- 96 9758 1360-1378 CGCUGUGUGGACCUCUUUGTsT 567CAAAGAGGUCCACACAGCGTsT 568 AD- 41 9567 1360-1378 cGcuGuGuGGAccucuuuGTsT569 cAAAGAGGUCcAcAcAGCGTsT 570 AD- 50 9693 1390-1408GACAUCAUUGGUGCCUCCATsT 571 UGGAGGCACCAAUGAUGUCTsT 572 AD- 81 104 95861390-1408 GAcAucAuuGGuGccuccATsT 573 UGGAGGcACcAAUGAUGUCTsT 574 AD- 1079712 1394-1412 UCAUUGGUGCCUCCAGCGATsT 575 UCGCUGGAGGCACCAAUGATsT 576 AD-120 9564 1394-1412 ucAuuGGuGccuccAGcGATsT 577 UCGCUGGAGGcACcAAUGATsT 578AD- 92 9690 1417-1435 AGCACCUGCUUUGUGUCACTsT 579 GUGACACAAAGCAGGUGCUTsT580 AD- 74 84 9616 1417-1435 AGcAccuGcuuuGcGucAcTsT 581GUGAcAcAAAGcAGGUGCUTsT 582 AD- 127 9742 1433-1451 CACAGAGUGGGACAUCACATT583 UGUGAUGUCCCACUCUGUGTT 584 AD- 24 15398 1486-1504AUGCUGUCUGCCGAGCCGGTsT 585 CCGGCUCGGCAGACAGCAUTsT 586 AD- 111 96171486-1504 AuGcuGucuGccGAGccGGTsT 587 CCGGCUCGGcAGAcAGcAUTsT 588 AD- 1049743 1491-1509 GUCUGCCGAGCCGGAGCUCTsT 589 GAGCUCCGGCUCGGCAGACTsT 590 AD-73 90 9635 1491-1509 GucuGccGAGccGGAGcucTsT 591 GAGCUCCGGCUCGGcAGACTsT592 AD- 83 9761 1521-1539 GUUGAGGCAGAGACUGAUCTsT 593GAUCAGUCUCUGCCUCAACTsT 594 AD- 76 9568 1521-1539 GuuGAGGcAGAGAcuGAucTsT595 GAUcAGUCUCUGCCUcAACTsT 596 AD- 52 9694 1527-1545GCAGAGACUGAUCCACUUCTsT 597 GAAGUGGAUCAGUCUCUGCTsT 598 AD- 47 95761527-1545 GcAGAGAcuGAuucAcuucTsT 599 GAAGUGGAUcAGUCUCUGCTsT 600 AD- 799702 1529-1547 AGAGACUGAUCCACUUCUCTsT 601 GAGAAGUGGAUCAGUCUCGTsT 602 AD-69 9627 1529-1547 AGAGAcuGAuccAcuucucTsT 603 GAGAAGUGGAUcAGUCUCUTsT 604AD- 127 9753 1543-1561 UUCUCUGCCAAAGAUGUCATsT 605 UGACAUCUUUGGCAGAGAATsT606 AD- 141 9628 1543-1561 uucucuGccAAAGAuGucATsT 607UGAcAUCUUUGGcAGAGAATsT 608 AD- 89 9754 1545-1563 CUCUGCCAAAGAUGUCAUCTsT609 GAUGACAUCUUUGGCAGAGTsT 610 AD- 80 9631 1545-1563cucuGccAAAGAuGucAucTsT 611 GAUGAcAUCUUUGGcAGAGTsT 612 AD- 78 97571580-1598 CUGAGGACCAGCGGGUACUTsT 613 AGUACCCGCUGGUCCUCAGTsT 614 AD- 3132 9595 1580-1598 cuGAGGAccAGcGGGuAcuTsT 615 AGuACCCGCUGGUCCUcAGTsT 616AD- 87 70 9721 1581-1599 UGAGGACCAGCGGGUACUGTsT 617CAGUACCCGCUGGUCCUCATsT 618 AD- 68 9544 1581-1599 uGAGGAccAGcGGGuAcUGTsT619 cAGuACCCGCUGGGCCUcATsT 620 AD- 67 9670 1666-1684ACUGUAUGGUCAGCACACUTT 621 AGUGUGCUGACCAUACAGUTT 622 AD- 25 152351668-1686 UGUAGGGUCAGCACACUCGTT 623 CGAGUGUGCCGACCAUACATT 624 AD- 7315236 1669-1687 GUAUGGUCAGCACACUCGGTT 625 CCGAGUGUGCUGACCAUACTT 626 AD-100 15168 1697-1715 GGAUGGCCACAGCCGUCGCTT 627 GCGACGGCUGUGGCCAUCCTT 628AD- 92 15174 1698-1716 GAUGGCCACAGCCGUCGCCTT 629 GGCGACGGCUGUGGCCAUCTT630 AD- 81 15235 1806-1824 CAAGCUGGUCUGCCGGGCCTT 631GGCCCGGCAGACCAGCUUGTT 632 AD- 65 15326 1815-1833 CUGCCGGGCCCACAACGCUTsT633 AGCGUGUGGGCCCGGCAGTsT 634 AD- 35 42 9570 1815-1833cuGccGGGcccAcAAcGcuTsT 635 AGCGUUGUGGGCCCGGcAGTsT 636 AD- 77 96961816-1834 UGCCGGGCCCACAACGCUUTsT 637 AAGCGUUGUGGGCCCGGCATsT 638 AD- 389566 1816-1834 uGccGGGcccAcAAcGcuuTsT 639 AAGCGUUGUGGGCCCGGcATsT 640 AD-78 9692 1818-1836 CCGGGCCCACAACGCUUUUTsT 641 AAAAGCGUUGUGGGCCCGGTsT 642AD- 100 9532 1818-1836 ccGGGcccAcAAcGccuuuTsT 643 AAAAGCGUUGUGGGCCCGGTsT644 AD- 102 9658 1820-1838 GCGGCCCACAACGCUUUUGTsT 645CCAAAAGCGUUGUGGGCCCTsT 646 AD- 50 9549 1820-1838 GGGcccAcAAcGcuuuuGGTsT647 CcAAAAGCGUUGUGGGCCCTsT 648 AD- 78 9675 1840-1858GGUGAGGGUGUCUACGCCATsT 649 UGGCGUAGACACCCUCACCTsT 650 AD- 43 95411840-1858 GGuGAGGGuGucuAcGccATsT 651 UGGCGuAGAcACCCUcACCTsT 652 AD- 739667 1843-1861 GAGGGUGUCUACGCCAUUGTsT 653 CAAUGGCGUAGACACCCUCTsT 654 AD-36 9550 1843-1861 GAGGGuGucuAcGccAuuGTsT 655 cAAUGGCGuAGAcACCCUCTsT 656AD- 100 9676 1861-1879 GCCAGGUGCUGCCUGCUACTsT 657 GUAGCAGGCAGCACCUGGCTsT658 AD- 27 32 9571 1861-1879 GccAGGuGcuGccUGcuAcTsT 659GuAGcAGGcAGcACCUGGCTsT 660 AD- 74 89 9697 1862-1880CCAGGUGCUGCCUGCUACCTsT 661 GGUAGCAGGCAGCACCUGGTsT 662 AD- 47 53 95721862-1880 ccAGGuGcuGccuGcuAccTsT 663 GGuAGcAGGcAGcACCUGGTsT 664 AD- 739698 2008-2026 ACCCACAAGCCGCCUGUGCTT 665 GCACAGGCGGCUUGUGGGUTT 666 AD-82 15327 2023-2041 GUGCUGAGGCCACGAGGUCTsT 667 GACCUCGUGGCCUCAGCACTsT 668AD- 30 35 9639 2023-2041 GuGcuGAGGccAcGAGGucTsT 669GACCUCGUGGCCUcAGcACTsT 670 AD- 82 74 9765 2024-2042UGCUGAGGCCACGAGGUCATsT 671 UGACCUCGUGGCCUCAGCATsT 672 AD- 31 35 0.609518 2024-2042 UGCUGAGGCCACGAGGUCATsT 673 UGACCUCGUGGCCUCAGCATsT 674 AD-31 9518 2024-2042 uGcuGAGGccAcGAGGucATsT 675 uGACCUCGUGGCCUcAGcATsT 676AD- 35 37 2.60 9644 2024-2042 UfgCfuGfaGfgCfcAfcGfaGfgUfCAfTsT 677p-aGfaCfcUfcGfuGfgCfcUfcAfgCfaTsT 678 AD- 26 14672 2024-2042UfGCfUfGAGGCfCfACfGAGGUfCfATsT 679 UfGACfCfUfCfGCfGGCfCfUfCfAGCfATsT 680AD- 27 15682 2024-2042 UgCuGaGgCcAcGaGgUcATsT 681p-uGfuCfcUfcGfuGfgCfcUfcAfgCfaTsT 682 AD- 22 14692 2024-2042UgCuGaGgCcAcGuGgUcATsT 683 GfGACfCfUfCfGUfGGCfCfUfCfAGCfATsT 684 AD- 1914702 2024-2042 UfgCfuGfaGfgCfcAfcGfaGfgUfcAfTsT 685UGACCucGUggCCUCAgcaTsT 686 AD- 25 14712 2024-2042UfGCfUfGAGGCfCfACfGAGGUfCfATsT 687 UGACCucGUggCCUCAgcaTsT 688 AD- 1814722 2024-2042 UgCuGaGgCcAcGuGgUcATsT 689 UGACCucGUggCCUCAgcaTsT 690AD- 32 14732 2024-2042 GfuGfgUfcAfgCfgGfcCfgGfgAfuGfTsT 691p-cAfuCfcCfgGgcCfgCfcGfaCfcAfcTsT 692 AD- 86 15078 2024-2042GUfGGUfCfAGCfGGCfCfGGGAUfGTsT 693 CfAUfCfCfCfGGCfCfGCfUfGACfGTACfTsT 694AD- 97 15088 2024-2042 GuGgUcAgCgGcCgGgAuGTsT 695p-cAfuCfcCfGGfcCfgCfuGfaCfcAfcTsT 696 AD- 74 15098 2024-2042GuGgUcAgCgGcCgGgAuGTsT 697 CfAUfCfCfCfGGCfCfGCfUfGACfCfACfTsT 698 AD- 6715108 2024-2042 GfuGfgUfCAfgCfgGfcCfgGfgAfuGfTsT 699CAUCCcgGCcgCUGACcacTsT 700 AD- 76 15118 2024-2042GUfGGUfCfAGCfGGCfCfGGGAUfGTsT 701 CAUCCcgGCcgCUGACcacTsT 702 AD- 8615128 2024-2042 GuGgUcAgCgGcCgGgAuGTsT 703 CAUCCcgGCcgCUGACcacTsT 704AD- 74 15138 2030-2048 GGCCACGAGGUCAGCCCAATT 705 UCGGGCUGACCUCGUGGCCTT706 AD- 30 15237 2035-2053 CGAGGUCAGCCCAACCAGUTT 707ACUGGUUGGGCUGACCUCGTT 708 AD- 30 15287 2039-2057 GUCAGCCCAACCAGUGCGUTT709 ACGCACUGGUUGGGCUGACTT 710 AD- 36 15238 2041-2059CAGCCCAACCAGUGCGUGGTT 711 CCACGCACUGGUUGGGCUGTT 712 AD- 35 152382062-2080 CACAGGGAGGCCAGCAUCCTT 713 GGAUGCUGGCCUCCCUGUGTT 714 AD- 4715399 2072-2090 CCAGCAUCCACGCUUCCUGTsT 715 CAGGAAGCGUGGAUGCUGGTsT 716AD- 37 9582 2072-2090 cCAGcAuccAcGcucccuGTsT 717 cAGGAAGCGGGGAUGCGGGTsT718 AD- 81 9708 2118-2136 AGUCAAGGAGCAUGGAAUCTsT 719GAUUCCAUGCUCCUUGACUTsT 720 AD- 31 43 9545 2118-2136AGucAAGGAGcAuGGAAccTsT 721 GAUUCcAUGCUCCUUGACUTsT 722 AD- 15 33 2.509671 2118-2136 AfgUfcAfaGfgAfgCfaUfgGfaAfuCfTsT 723p-gAfuUfcCfuUfgCfaCfcUfuGfaCfuTsT 724 AD- 16 14674 2118-2136AGUfCfAAGGAGCfAUfGGAAUfCfTsT 725 GAUfUfCfCfAUfGCfUfCfCfUfGfGACfUfTs 726AD- 26 T 14684 2118-2136 AgUcAaGgAgCaUgGaAcCTsT 727p-gAfuGfcCfaUfgCfuCfcUfuGfuCfuTsT 728 AD- 18 14694 2118-2136AgUcAaGgAgCaUgGuAuCTsT 729 GAUfUfCfCfAUfGCfUfCfCfUfUfGACfUFTs 730 AD- 27T 14704 2118-2136 AfgUfcAfuGfgAfgCfaUfgGgaAfcCfTsT 731GAUUCcaUGcuCCUUGacuTsT 732 AD- 20 14714 2118-2136AGUfCfAAGGAGCfAUfGGAAUfCTTsT 733 GAUUCcaUGcuCCUUGucuTsT 734 AD- 18 147242118-2136 AgUcAaGgAgCaUgGaAuCTsT 735 GAUUCcaUGcuCCUUGucuTsT 736 AD- 1814734 2118-2136 GfcGfgCfuCfcCfuCfaUfaGfgCfcUfTsT 737p-aGfgCfcUfaUfgAfgGfgUfgCfcGfcTsT 738 AD- 29 15080 2118-2136GCfGGCfACfCfCfUfCfAUfAGGCfCfUfTsT 739 AGGCfCfUfAUfGAGGGUfGCfCfGCfTsT 740AD- 23 15090 2118-2136 GcGgCaCcCuCaUaGgCcUTsT 741p-aGfgCfcUfaUfgAfgGfgUfgCfcGfcTsT 742 AD- 26 15100 2118-2316GcGgCaCcCuCaUaGgCcUTsT 743 AGGCfCfGfAUfGAGGGUfGCfCfCCfTsT 744 AD- 2315110 2118-2136 GfcGfgCfaCfcCfuCfaUfaGfgCfcUfTsT 745AGGCCuaUGagGGUGCcgcTsT 746 AD- 20 15120 2118-2136GCfGGCfACfCfCfUfCfAUfAGGCfCfUfTsT 747 AGGCCuuUGugGGUGCcgcTsT 748 AD- 2015130 2118-2136 GcGgCaCcCuCaUaGgCcUTsT 749 AGGCCuaUGagGGUGCcgcTsT 750AD- 19 15140 2122-2140 AAGGAGCAUGGAAUCCCGGTsT 751 CCGGGAUUCCAUGCUCCUUTsT752 AD- 59 9522 2122-2140 AAGGAGcAuGGAAucccGGTsT 753CCGGGAUUCcAUGCUCCUUTsT 754 AD- 78 9648 2123-2141 AGGAGCAUGGAAUCCCGGCTsT755 GCCGGGAUUCCAUGCUCCUTsT 756 AD- 80 9552 2123-2141AGGAGcAuGGAAucccGGcTsT 757 GCCGGGAUUCcAUGCUCCUTsT 758 AD- 76 96782125-2143 GAGCAUGGAAUCCCGGCCCTsT 759 GGGCCGGGAUUCCAUGCUCTsT 760 AD- 909618 2125-2143 GAGcAuGGAAucccGGcccTsT 761 GGGCCGGGAUUCcAUGCUCTsT 762 AD-91 9744 2230-2248 GCCUACGCCGUAGACAACATT 763 UGUUGUCUACGGCGUAGGCTT 764AD- 38 15239 2231-2249 CCCACGCCGUAGACAACACTT 765 GUGUUGUCUACGGCGUAGGTT766 AD- 19 15212 2232-2250 CUACGCCGUAGACAACACGTT 767CGUGUUGUCUACGGCGUAGTT 768 AD- 43 15240 2233-2251 UACGCCGUAGACAACACGUTT769 ACGUGUUGUCUACGGCGUATT 770 AD- 59 15177 2235-2253CGCCGUAGACAACACGUGUTT 771 ACACGUGUUGUCUACGGCGTT 772 AD- 13 151792236-2254 GCCGUAGACAACACGUGUGTT 773 CACACGUGUUGUCUACGGCTT 774 AD- 1515180 2237-2255 CCGUAGACAACACGUGUGUTT 775 ACACACGUGUUGUCUACGGTT 776 AD-14 15241 2238-2256 CGUAGACAACACGUGUGUATT 777 UACACACGUGUUGUCUACGTT 778AD- 42 15268 2240-2258 UAGACAACACGUGUGUAGUTT 779 ACUACACACGUGUUGUCUATT780 AD- 21 15242 2341-2259 AGACAACACGUGUGUAGUCTT 781GACUACACACGUGUUGUCUTT 782 AD- 28 15216 2242-2260 GACAACACGUGUGUAGUCATT783 UGACUACACACGUGUUGUCTT 784 AD- 35 15176 2243-2261ACAACACGUGUGUAGUCAGTT 785 CUGACUACACACGUGUUGUTT 786 AD- 35 151812244-2262 CAACACGUGUGUAGUCAGGTT 787 CCUGACUACACACGUGUUGTT 788 AD- 2215243 2247-2265 CACGUGUGUAGUCAGGAGCTT 789 GCUCCUGACUACACACGUGTT 790 AD-42 15182 2348-2266 ACGUGUGUAGUCAGGAGCCTT 791 GGCUCCUGACUACACACGUTT 792AD- 31 15244 2249-2267 CGUGUGUAGUCAGGAGCCGTT 793 CGGCUCCUGACUACACACGTT794 AD- 23 15387 2251-2269 UGUGUAGUCAGGAGCCGGGTT 795CCCGGCUCCUGACUACACATT 796 AD- 18 15245 2257-2275 GUCAGGAGCCGGGACGUCATsT797 UGACGUCCCGGCUCCUGACTsT 798 AD- 34 9555 2257-2275GucAGGAGccGGGAcGucATsT 799 UGACGUCCCGGCUCCUGACTsT 800 AD- 55 96812258-2276 UCAGGAGCCGGGACGUCAGTsT 801 CUGACGUCCCGGCUCCUGATsT 802 AD- 4261 9619 2258-2276 ucAGGAGccGGGAcGucAGTsT 803 CUGACGUCCCGGCUCCUGATsT 804AD- 56 9745 2259-2277 CAGGAGCCGGGACGUCAGCTsT 805 GCUGACGUCCGGGCUCCUGTsT806 AD- 44 77 9620 2259-2277 cAGGAGccGGGAcGucAGcTsT 807GCUGACGUCCCGGCUCCUGTsT 808 AD- 89 9746 2263-2281 AGCCGGGACGUCAGCACUATT809 UAGUGCUGACGUCCCGGCUTT 810 AD- 19 15288 2265-2283CCGGGACGUCAGCACUACATT 811 UGUAGUGCUGACGUCCCGGTT 812 AD- 16 152462303-2321 CCGUGACAGCCGUUGCCAUTT 813 AUGGCAACGGCUGUCACGGTT 814 AD- 3715289 2317-2335 GCCAUCUGCUGCCGGAGCCTsT 815 GGCUCCGGCAGCAGAUGGCTsT 816AD- 59 67 9324 2375-2393 CCCAUCCCAGGAUGGGUGUTT 817 ACACCCAUCCUGGGAUGGGTT818 AD- 103 15329 2377-2395 CAUCCCAGGAUGGGUGUCUTT 819AGACACCCAUCCUGGGAUGTT 820 AD- 62 15330 2420-2438 AGCUUUAAAAUGGUUCCGATT821 UCGGAACCAUUUUAAAGCUTT 822 AD- 22 15169 2421-2439GCUUUAAAAUGGUUCCGACTT 823 GUCGGAACCAUUUUAAAGCTT 824 AD- 6 152012422-2440 CUUUAAAAUGGUUCCGACUTT 825 AGUCGGAACCAUUUUAAAGTT 826 AD- 1415331 2423-2441 UUUAAAAUGGUUCCGACUUTT 827 AAGUCGGAACCAUUUUAAATT 828 AD-47 15190 2424-2442 UUAAAAUGGUUCCGACUUGTT 829 CAAGUCGGAACCAUUUUAATT 830AD- 61 15247 2425-2443 UAAAAUGGUUCCGACUUGUTT 831 ACAAGUCGGAACCAUUUUATT832 AD- 22 15248 2426-2444 AAAAUGGUUCCGACUUGUCTT 833GACAAGUCGGAACCAUUUUTT 834 AD- 45 15175 2477-2445 AAAUGGUUCCGACUUGUCCTT835 GGACAAGUCGGAACCAUUUTT 836 AD- 51 15249 2428-2446AAUGGUUCCGACUUGUCCCTT 837 GGGACAAGUCGGAACCAUUTT 838 AD- 96 152502431-2449 GGUUCCGACUUGUCCCUCUTT 839 AGAGGGACAAGUCGGAACCTT 840 AD- 1215400 2457-2475 CUCCAUGGCCUGGCACGAGTT 841 CUCGUGCCAGGCCAUGGAGTT 842 AD-22 15332 2459-2477 CCAUGGCCUGGCACGAGGGTT 843 CCCUCGUGCCAGGCCAUGGTT 844AD- 30 15388 2545-2563 GAACUCACUCACUCUGGGUTT 845 ACCCAGAGUGAGUGAGUUCTT846 AD- 20 15333 2549-2567 UCACUCACUCUGGGUGCCUTT 847AGGCACCCAGAGUGAGUGATT 848 AD- 96 15334 2616-2634 UUUCACCAUUCAAACAGGUTT849 ACCUGUUUGAAUGGUGAAATT 850 AD- 75 15335 2622-2640CAUUCAAACAGGUCGAGCUTT 851 AGCUCGACCUGUUUGAAUGTT 852 AD- 16 151832623-2641 AUUCAAACAGGUCGAGCUGTT 853 CAGCUCGACCGUUUUGAAUTT 854 AD- 4115202 2624-2642 UCCAAACAGGUCGAGCUGUTT 855 ACAGCUCGACCUGUUUGAATT 856 AD-39 15203 2625-2643 UCAAACAGGUCGAGCUGUGTT 857 CACAGCUCGACCUGUUUGATT 858AD- 49 15272 2626-2644 CAAACAGGUCGAGCUGUGCTT 859 GCACAGCUCGACCUGUUUGTT860 AD- 16 15217 2627-2645 AAACAGGUCGAGCUGUGCUTT 861AGCACAGCUCGACCUGUUUTT 862 AD- 15 15290 2628-2546 AACAGGUCGAGCUGUGCUCTT863 GAGCACAGCUCGACCUGUUTT 864 AD- 13 15218 2630-2648CAGGUCGAGCUGUGCUCGGTT 865 CCGAGCACAGCUCGACCUGTT 866 AD- 13 153892631-2649 AGGUCGAGCUGUGCCCGGGTT 867 CCCGAGCACAGCUCGACCUTT 868 AD- 4015336 2633-2651 GUCGAGCUGUGCUCGGGUGTT 869 CACCCGAGCACAGCUCGACTT 870 AD-19 15337 2634-2652 UCGAGCUGUGCUCGGGUGCTT 871 GCACCCGAGCACAGCUCGATT 872AD- 33 15191 2657-2675 AGCUGCUCCCAAUGUGCCGTT 873 CGGCACAUUGGGAGCAGCUTT874 AD- 25 15390 2658-2676 GCUGCUCCCAAUGUGCCGATT 875UCGGCACAUUGGGAGCAGCTT 876 AD- 9 15338 2660-2678 UGCUCCCAAUGUGCCGAUGTT877 CAUCGGCACAUUGGGAGCATT 878 AD- 33 15204 2663-2681UCCCAAUGUGCCGAUGUCCTT 879 GGACAUCGGCACAUUGGGATT 880 AD- 76 152512665-2683 CCAAUGUGCCGAUGUCCGUTT 881 ACGGACAUCGGCACAUUGGTT 882 AD- 1415205 2666-2684 CAAUGUGCCGAUGUCCGUGTT 883 CACGGACAUCGGCACAUUGTT 884 AD-16 15171 2667-2683 AAUGUGCCGAUGUCCGUGGTT 885 CCACGGACAUCGGCACAUUTT 886AD- 58 15252 2673-2691 CCGAUGUCCGUGGGCAGAATT 887 UUCUGCCCACGGACAUCGGTT888 AD- 20 15339 2675-2693 GAUGUCCGUGGGCAGAAUGTT 889CAUUCUGCCCACGGACAUCTT 890 AD- 15 15253 2678-2696 GUCCGUGGGCAGAAUGACUTT891 AGUCAUUCUGCCCACGGACTT 892 AD- 18 15340 2679-2697UCCGUGGGCAGAAUGACUUTT 893 AAGUCAUUCUGCCCACGGATT 894 AD- 17 152912683-2701 UGGGCAGAAUGACUUUUAUTT 895 AUAAAAGUCAUUCUGCCCATT 896 AD- 1115341 2694-2712 ACUUUUAUUGAGCUCUUGUTT 897 ACAAGAGCUCAAUAAAAGUTT 898 AD-13 15401 2700-2718 AUUGAGCUCUUGUUCCGUGTT 899 CACGGAACAAGAGCUCAAUTT 900AD- 30 15342 2704-2722 AGCUCUUGUUCCGUGCCAGTT 901 CUGGCACGGAACAAGAGCUTT902 AD- 21 15343 2705-2723 GCUCUUGUUCCGUGCCAGGTT 903CCUGGCACGGAACAAGAGCTT 904 AD- 16 15292 2710-2728 UGUUCCGUGCCAGGCAUUCTT905 GAAUGCCUGGCACGGAACATT 906 AD- 20 15344 2711-2729GUUCCGUGCCAGGCAUUCATT 907 UGAAUGCCUGGCACGGAACTT 908 AD- 18 152542712-2730 UUCCGUGCCAGGCAUUCAATT 909 UUGAAUGCCUGGCACGGAATT 910 AD- 1815345 2715-2733 CGUGCCAGGCAUUCAAUCCTT 911 GGAUUGAAUGCCUGGCACGTT 912 AD-15 15206 2716-2734 GUGCCAGGCAUUCAAUCCUTT 913 AGGAUUGAAUGCCUGGCACTT 914AD- 16 15346 2728-2746 CAAUCCUCAGGUCUCCACCTT 915 GGUGGAGACCUGAGGAUUGTT916 AD- 62 15347 2743-2761 CACCAAGGAGGCAGGAUUCTsT 917GAAUCCUGCCUCCUUGGUGTsT 918 AD- 33 31 9577 2743-2761cAccAAGGAGGcAGGAuucTsT 919 GAAUCCUGCCUCCUUGGUGTsT 920 AD- 17 26 97032743-2761 CfaCfcAfaGfgAfgGfcAfgGfaCfuCfTsT 921p-gAfaUfcDfuGfcCfuCfcUfuGfgUfgTsT 922 AD- 22 14678 2743-2761CfACfCfAAGGAGGCfAGGAUfCfCfTsT 923 GAAUfCfCfUfGCfCfUfCfCfUfUfGGUfGTs 924AD- 23 T 14688 2743-2761 CuCcAaGgAgGcAgGaUuCTsT 925p-gAfaUfcCfuGfcCfuCfcUfuGfgUfgTsT 926 AD- 23 14698 2743-2761CaCcAuGgAgGcAgGuUuCTsT 927 GAAUfCfCfGfGCfCfUfCfCfUfUfGTs 928 AD- 14 T14708 2743-2761 CfaCfcAfaGfgAfgGfcAfgGfaUfuCfTsT 929GAAUCcuGCcuCCUUCgcgTsT 930 AD- 31 14718 2743-2761CfACfCfAAGGAGGCfAGGAUfUfCfTsT 931 GAAUCcuGCcuCCUUGgcgTsT 932 AD- 2514728 2743-2761 CaCcAuGgAgGcAgGaUuCTsT 933 GAAUCcuGCcUCCUUGgugTsT 934AD- 31 14738 2743-2761 GfgCfcUfGGfaGfuAfuUfcGfgAfTsT 935p-uCfcGfaAfuAfaAfcUfcCfaGfgCfcTsT 936 AD- 19 15084 2743-2761GGCfCfUfGGAGUfUfUfAUfUfCfGGATsT 937 UfCfCfGAAUfAAACfUfCfCfAGGCfCfTsT 938AD- 31 15094 2743-2761 GgCcuGGaGuUuAuUcGgATsT 939p-a-CfcGfaAfuAfaAfcUfcCfuGfgCfcTsT 940 AD- 16 15104 2743-2761GgCcUgGaGuUuAuUcGgATsT 941 UfCfCfGAAUfAAACfUfCfCfAGGCfCfTsT 942 AD- 1515114 2743-2761 GfgCfcUfgGfaGfuUfuAfuUfcGfgAfTsT 943UCCGAauAAacUCCAGgCCTsT 944 AD- 11 15124 2743-2761CCCfCfUfGGAGUfUfUfAUfUfCfGGATsT 945 UCCGAauAAacUCCAGgccTsT 946 AD- 1215134 2743-2761 GgCcUgGaGuUuAuUcGgATsT 947 UCCGAauAAucUCCAGgccTsT 948AD- 9 15144 2753-2771 GCAGGAUUCUUCCCAUGGATT 949 UCCAUGGGAAGAAUCCUGCTT950 AD- 7 15391 2794-2812 UGCAGGGACAAACAUCGUUTT 951AACGAUGUUUGUCCCUGCATT 952 AD- 13 15348 2795-2813 GCAGGGACAAACAUCGUUGTT953 CAACGAUGUUUGUCCCUGCTT 954 AD- 8 15349 2798-2815AGGGACAAACAUCGUUGGGTT 955 CCCAACGAUGUUUGUCCCUTT 956 AD- 40 151702841-2859 CCCUCAUCUCCAGCUAACUTT 957 AGUUAGCUGGAGAUGAGGGTT 958 AD- 1415350 2845-2863 CAUCUCCAGCUAACUGUGGTT 959 CCACAGUUAGCUGGAGAUGTT 960 AD-27 15402 2878-2896 GCUCCCUGAUUAAUGGAGGTT 961 CCUCCAUUAAUCAGGGAGCTT 962AD- 27 15293 2881-2899 CCCUGAUUAAUGGAGGCUUTT 963 AAGCCGCCAUUAAUCAGGGTT964 AD- 14 15351 2882-2900 CCUGAUUAAUGGAGGCUUATT 965UAAGCCUCCAUUAAUCAGGTT 966 AD- 11 15403 2884-2902 UGAUUAAUGGAGGCUUAGCTT967 GCUAAGCCUCCAUUAAUCATT 968 AD- 38 15404 2885-2903GAUUAAUGGAGGCUUAGCUTT 969 AGCUAAGCCUCCAUUAAUCTT 970 AD- 15 152072886-2904 AUUAAUGGAGGCUUAGCUUTT 971 AAGCUAAGCCUCCAGUAAUTT 972 AD- 2315352 2887-2905 UUAAUGGAGGCUUAGCUUUTT 973 AAAGCCAAGCCUCCAUUAATT 974 AD-31 15255 2903-2921 UUUCUGGAUGGCAUCUAGCTsT 975 GCUAGAUGCCAUCCAGAAATsT 976AD- 123 9603 2903-2921 uuacuGGAuGGcAucuAGcTsT 977 GCuAGAUGCcAUCcAGAAATsT978 AD- 56 9729 2904-2922 UUCUGCAUGGCAUCUAGCCTsT 979GGCUAGAUGCCAUCCAGAATsT 980 AD- 139 9599 2904-2922 uucuGGAuGGcAucuAGccTsT981 GGCuAGAUGCcAUCcAGAATsT 982 AD- 38 9725 2905-2923UCUGGAUGGCAUCUAGCCATsT 983 UGGCUAGAUGCCAUCCAGATsT 984 AD- 77 96212905-2923 ucuGGAuGGcAucuAGccATsT 985 UGGCuAGAUGCcAUCcAGATsT 986 AD- 639747 2925-2943 AGGCUGGAGACAGGUGCGCTT 987 GCGCACCUGUCUCCAGCCUTT 988 AD-32 15405 2926-2944 GGCUGGAGACAGGUGCGCCTT 989 GGCGCACCUGUCUCCAGCCTT 990AD- 39 15353 2927-2945 GCUGGAGACAGGUGCGCCCTT 991 GGGCGCACCUGUCUCCAGCTT992 AD- 49 15354 2972-2990 UUCCUGAGCCACCUUUACUTT 993AGUAAAGGUGGCUCAGGAATT 994 AD- 35 15406 2973-2991 UCCUGAGCCACCUUUACUCTT995 GAGUAAAGGUGGCUCAGGATT 996 AD- 39 15407 2974-2992CCUGAGCCACCUUUACUCUTT 997 AGAGUAAAGGUGGCUCAGGTT 998 AD- 18 153552976-2994 UGAGCCACCUUUACUCUGCTT 999 GCAGAGUAAAGGUGGCUCATT 1000 AD- 5015356 2978-2996 AGCCACCUUUACUCUGCUCTT 1001 GAGCAGAGUAAAGGUGGCUTT 1002AD- 54 15357 2981-2999 CACCUUUACUCUGCUCUAUTT 1003 AUAGAGCAGAGUAAAGGUGTT1004 AD- 23 15269 2987-3005 UACUCUGCUCUAUGCCAGGTsT 1005CCUGGCAUAGAGCAGAGUATsT 1006 AD- 74 9565 2987-3005 uAcucuGcucuAuGccAGGTsT1007 CCUGGcAuAGAGcAGAGuATsT 1008 AD- 49 9691 2998-3016AUGCCAGGCUGUGCUAGCATT 1009 UGCUAGCACAGCCUGGCAUTT 1010 AD- 12 15358303-3021 AGGCUGUGCUAGCAACACCTT 1011 GGUGUUGCUAGCACAGCCUTT 1012 AD- 2415359 3006-3024 CUGUGCUAGCAACACCCAATT 1013 UUGGGUGUUGCUAGCACAGTT 1014AD- 13 15360 3010-3028 GCUAGCAACACCCAAAGGUTT 1015 ACCUUUGGGUGUUGCUAGCTT1016 AD- 19 15219 3038-3056 GGAGCCAUCACCUAGGACUTT 1017AGUCCUAGGUGAUGGCUCCTT 1018 AD- 24 15361 3046-3064 CACCUAGGACUGACUCGGCTT1019 GCCGAGUCAGUCCUAGGUGTT 1020 AD- 36 15273 3051-3069AGGACUGACUCGGCAGUGUTT 1021 ACACUGCCGAGUCAGUCCGTT 1022 AD- 31 153623052-3070 GGACUGACUCGGCAGUGUGTT 1023 CACACUGCCGAGCCAGUCCTT 1024 AD- 2015192 3074-3092 UGGUGCAUGCACUGUCUCATT 1025 UGAGACAGUGCAUGCACCATT 1026AD- 19 15256 3080-3098 AUGCACUGUCUCAGCCAACTT 1027 GUUGGCUGAGACAGUGCAUTT1028 AD- 33 15363 3085-3103 CUGUCUCAGCCAACCCGCUTT 1029AGCGGGUUGGCUGAGACAGTT 1030 AD- 24 15364 3089-3107 CUCAGCCAACCCGCUCCACTsT1031 GUGGAGCGGGUUGGCUGAGTsT 1032 AD- 35 49 9604 3089-3107cucAGccAAcccGcuccAcTsT 1033 GUGGAGCGGGUUGGCUGAGTsT 1034 AD- 85 97303093-3111 GCCAACCCGCUCCACUACCTsT 1035 GGUAGUGGAGCGGGUUGGCTsT 1036 AD- 459527 3093-3111 GccAAcccGcuccAcuAccTsT 1037 GGuAGUGGAGCGGGUUGGCTsT 1038AD- 86 9653 3096-3114 AACCCGCUCCACUACCCGGTT 1039 CCGGGUAGUGGAGCGGGUUTT1040 AD- 62 15365 3099-3117 CCGCUCCACUACCCGGCAGTT 1041CUGCCGGGUAGUGGAGCGGTT 1042 AD- 30 15294 3107-3125 CUACCCGGCAGGGUACACATT1043 UGUGUACCCUGCCGGGUAGTT 1044 AD- 12 15173 3108-3126UACCCGGCAGGGUACACAUTT 1045 AUGUGUACCCUGCCGGGUATT 1046 AD- 21 153663109-3127 ACCCGGCAGGGUACACAUUTT 1047 AAUGUGUACCCUGCCGGGUTT 1048 AD- 1115367 3110-3128 CCCGGCAGGGUACACAUUCTT 1049 GAAUGUGUACCCUGCCGGGTT 1050AD- 18 15257 3112-3139 CGGCAGGGUACACAUUCGCTT 1051 GCGAAUGUGUACCCUGCCGTT1052 AD- 50 15184 3114-3132 GCAGGGUACACAUUCGCACTT 1053GUGCGAAUGUGUACCCUGCTT 1054 AD- 12 15185 3115-3133 CAGGGUACACAUUCGCACCTT1055 GGUGCGAAUGUGUACCCUGTT 1056 AD- 73 15258 3116-3134AGGGUACACAUUCGCACCCTT 1057 GGGUGCGAAUGUGUACCCUTT 1058 AD- 36 151863196-3214 GGAACUGAGCCAGAAACGCTT 1059 GCGUUUCUGGCUCAGUUCCTT 1060 AD- 1915274 3197-3215 GAACUGAGCCAGAAACGCATT 1061 UGCGUUUCUGGCUCAGUUCTT 1062AD- 7 15368 3198-3216 AACUGAGCCAGAAACGCAGTT 1063 CUGCGUUUCUGGCUCAGUUTT1064 AD- 17 15369 3201-3219 UGACCCAGAAACGCAGAUUTT 1065AAUCUGCGUUUCUGGCUCATT 1066 AD- 19 15370 3207-3225 AGAAACGCAGAUUGGGCUGTT1067 CAGCCCAAUCUGCGUUUCUTT 1068 AD- 38 15259 3210-3228AACGCAGAUUGGGCUGGCUTT 1069 AGCCAGCCCAAUCUGCGUUTT 1070 AD- 52 154083233-3251 AGCCAAGCCUCUUCUUACUTsT 1071 AGUAAGAAGAGGCUUGGCUTsT 1072 AD- 2321 0.04 9597 3233-3251 AGccAAGccucuucuuAcuTsT 1073AGuAAGAAGAGGCUUGGCUTsT 1073 AD- 12 26 9723 3233-3251AfgCfcAfuGfcCfuCfuUfcUfuAfcUfTsT 1075 p-aGfuAfaGfaAfgAfgGfcUfuGfgCfuTsT1075 AD- 15 14680 3233-3251 AGCfCfAAGCfCfUfCfUfUfCfUfUfACfUfTsT 1077AGUfAAGAAGAGGCfUfUfGGCfUfTsT 1078 AD- 18 14690 3233-3251AgCcAaGcCuCuUcUuAcUTsT 1079 p-aGfuAfaGfaAfgAfgGfcUfcGfgCfuTsT 1080 AD-15 14700 3233-3251 AgCcAaGcCuCuUcUuAcUTsT 1081AGUfAAGAAGAGGCfUfCfGGCfUfTsT 1082 AD- 15 14710 3233-3251AfgCfcAfuGfcCfuCfuUfcUfuAfcUfTsT 1083 AGUAAgaAGagGCUUGgcuTsT 1084 AD- 1814720 3233-3251 AGCfCfAAGCfCfUfCfUfUfCfUfUfACfUfTsT 1085AGUAAgaAGaGGCUUGgcuTsT 1086 AD- 18 14730 3233-3251AgCcAaGcCuCuUcUuAcUTsT 1087 AGUAAgaAGagGCUUGgcaTsT 1088 AD- 17 147403233-3251 UfgGfuUfcCfcUfgAfgGfaCfcAfgCfTsT 1089p-gCfuGfgUfcCfuCfuGfgGfaAfcCfaTsT 1090 AD- 85 15086 3233-3251UfGGUfUfCfCfCfUfGAGGACfCfAGCfTsT 1091 GCfUfGGUfCfCfUfCfAGGGAACfCfATsT1092 AD- 70 15096 3233-3251 UgGuUcCcUgAgGaCcAgCTsT 1093p-gCfugFGuFcCfuCfaGfgGfaAfcCfaTsT 1094 AD- 71 15106 3233-3251UgGuUcCcUgAgGuCcAgCTsT 1095 GCfUfGGUfCfCfUfCfAGGGAACfCfATsT 1096 AD- 7315116 3233-3251 UfgGfuUfcCfcUfgAfgGfaCfcAfgCfTsT 1097GCUGGucCUcuGGGAAccaTsT 1098 AD- 71 15126 3233-3251UfGGUfUfCfCfCfUfGAGGACfCfAGCfTsT 1099 GCUGGucCUcaGGGAAccaTsT 1100 AD- 5615136 3233-3251 UgGuUcCcUgAgGaCcAgCTsT 1101 GCUGGucCUcaGGGAAccaTsT 1102AD- 72 15146 3242-3260 UCUUCUUACUUCACCCGGCTT 1103 GCCGGGUGAAGUAAGAAGATT1104 AD- 79 15260 3243-3261 CUUCUUACUUCACCCGGCUTT 1105AGCCGGGUGAAGUAAGAAGTT 1106 AD- 24 15371 3244-3262 UUCUUACUUCACCCGGCUGTT1107 CAGCCGGGUGAAGUAAGAATT 1108 AD- 52 15372 3262-3280GGGCUCCUCAUUUUUACGGTT 1109 CCGUAAAAAUGAGGAGCCCTT 1110 AD- 27 151723263-3281 GGCUCCUCAUUUUUACGGGTT 1111 CCCGUAAAAAUGAGGAGCCTT 1112 AD- 2215295 3264-3282 GCUCCUCAUUUUUACGGGUTT 1113 ACCCGUAAAAAUGAGGAGCTT 1114AD- 11 15373 3263-3283 CUCCUCAUUUUUACGGGUATT 1115 UACCCGUAAAAAUGAGGAGTT1116 AD- 18 15163 3266-3284 UCCUCAUUUUUACGGGUAATT 1117UUACCCGUAAAAAUGAGGATT 1118 AD- 13 15165 3267-3285 CCUCAUUUUUACGGGUAACTT1119 GUUACCCGGAAAAAUGAGGTT 1120 AD- 23 15374 3268-3286CUCAUUUUUACGGGUAACATT 1121 UGUUACCCGUAAAAAUGAGTT 1122 AD- 13 152963270-3288 CAUUUUUACGGGUAACAGUTT 1123 ACUGUUACCCGUAAAAAUGTT 1124 AD- 2015261 3271-3289 AUUUUUACGGGUAACAGUGTT 1125 CACUGUUACCCGUAAAAAUTT 1126AD- 90 15375 3274-3292 UUUACGGGUAACAGUGAGGTT 1127 CCUCACUGUUACCCGUAAATT1128 AD- 72 15262 3308-3326 CAGACCAGGAAGCUCGGUGTT 1129CACCGAGCUUCCUGGUCUGTT 1130 AD- 14 15376 3310-3328 GACCAGGAAGCUCGGUGAGTT1131 CUCACCGAGCUUCCUGGUCTT 1132 AD- 19 15377 3312-3330CCAGGAAGCUCGGUGAGUGTT 1133 CACUCACCGAGCUUCCUGGTT 1134 AD- 17 154093315-3333 GGAAGCUCGGUGAGUGAUGTT 1135 CAUCACUCACCGAGCUUCCTT 1136 AD- 1815378 3324-3342 GUGAGUGAUGGCAGAACGATT 1137 UCGUUCUGCCAUCACUCACTT 1138AD- 8 15410 3326-3344 GAGUGAUGGCAGAACGAUGTT 1139 CAUCGUUCUGCCAUCACUCTT1140 AD- 11 15379 3330-3348 GAUGGCAGAACGAUGCCUGTT 1141CAGGCAUCGUUCUGCCAUCTT 1142 AD- 36 15187 3336-3354 AGAACGAUGCCUGCAGGCATT1143 UGCCUGCAGGCAUCGUUCUTT 1144 AD- 18 15263 3339-3357ACGAUGCCUGCAGGCAUGGTT 1145 CCAUGCCUGCAGGCAUCGUTT 1146 AD- 75 152643348-3366 GCAGGCAUGGAACUUUUUCTT 1147 GAAAAAGUUCCAUGCCUGCTT 1148 AD- 2115297 3356-3374 GGAACUUUUUCCGUUAUCATT 1149 UGAUAACGGAAAAAGUUCCTT 1150AD- 6 15208 3357-3375 GAACUUUUUCCGUUAUCACTT 1151 GUGAGAACGGAAAAAGUUCTT1152 AD- 28 15209 3358-3376 AACUUUUUCCGUUAUCACCTT 1153GGUGAUAACGGAAAAAGUUTT 1154 AD- 131 15193 3370-3388 UAUCACCCAGGCCUGAUUCTT1155 GAAUCAGGCCUGGGUGAUATT 1156 AD- 88 15380 3378-3396AGGCCUGAUUCACUGGCCUTT 1157 AGGCCAGUGAAUCAGGCCUTT 1158 AD- 43 152983383-3401 UGAUUCACUGGCCUGGCGGTT 1159 CCGCCAGGCCAGUGAAUCATT 1160 AD- 9915299 3385-3403 AUUCACUGGCCUGGCGGAGTT 1161 CUCCGCCAGGCCAGUGAAUTT 1162AD- 95 15265 3406-3424 GCUUCUAAGGCAUGGUCGGTT 1163 CCGACCAUGCCUUAGAAGCTT1164 AD- 18 15381 3407-3425 CUUCUAAGGCAUGGUCGGGTT 1165CCCGACCAUGCCUUAGAAGTT 1166 AD- 40 15210 3429-3447 GAGGGCCAACAACUGUCCCTT1167 GGGACAGUCGUUGGCCCUCTT 1168 AD- 83 15270 3440-3458ACUGUCCCUCCUUGAGCACTsT 1169 GUGCUCAAGGAGGGACAGUTsT 1170 AD- 75 95 95913440-3458 AcuGucccuccuuGAGcAcTsT 1171 GUGCUcAAGGAGGGAcAGUTsT 1172 AD-105 9717 3441-3459 CUGUCCCUCCUUGAGCACCTsT 1173 GGUGCUCAAGGAGGGACAGTsT1174 AD- 94 9622 3441-3459 cuGucccuccuuGAGcAccTsT 1175GGUGCUcAAGGAGGGAcAGTsT 1176 AD- 103 9748 3480-3498ACAUUUAUCUUUUGGGUCUTsT 1177 AGACCAAAGAUAAAUGUTsT 1178 AD- 63 49 95873480-3498 AcAuuuAucuuuuGGGucuTsT 1179 AGACCcAAAAGAuAAAUGUTsT 1180 AD- 2225 9713 3480-3498 AfcAfuUfuAfuCfuUfuUfgGfgUfcUfTsT 1181p-aGfuCfcCfaAfaAfgAfuAfaAfuGfuTsT 1182 AD- 19 14679 3480-3498ACfAUfUfUfAUfCfUfUfCfUfGGGUfCfUfTs 1183 AGACfCfCfAAAAGAUfAAAfGUfTsT 1184AD- 24 T 14689 3480-3498 AcAuUuAuCaUuUgGgUcUTsT 1185p-aGfaCfcCfaAfaAfgAfuAfaAfuGfuTsT 1186 AD- 19 14699 3480-3498AcAuUuAuCcUuUgGgUcUTsT 1187 AGACfCfCfAAAAGAUfAAAUfGUTsT 1188 AD- 2114709 3480-3498 AfcAfuUfuAfuDfuUfuUfgGfgUfcUfTsT 1189AGACCcaAAagAUAAAuguTsT 1190 AD- 24 14719 3480-3498ACfAUfUfUfAUfCfUfUfCfUfGGGUfCfUfTs 1191 AGACCcaAAagAUAAAuguTsT 1192 AD-23 T 14729 3480-3498 AcAuUuAuCuUuUgGgUcUTsT 1193 AGACCcaAAagAUAAAuguTsT1194 AD- 24 14739 3480-3498 GfcCfaUfcUfgCfuGfcCfgGfaGfcCfTsT 1195p-gGfcUfcCfgGfcAfgCfuGfaUfggfcTsT 1196 AD- 74 15085 3480-3498GCfCfAUfCfUfGCfUfGCfCfGGAGCfCfTsT 1197 GGCfUfCfCfGGCTAGCTAGAUfGGCfTsT1198 AD- 60 15095 3480-3498 GcCaUcGgCuGcCgGaGcCTsT 1199p-gGfcUfcCfgGfcAfgCfuGfaUfggfcTsT 1200 AD- 33 15105 3480-3498GcCaUcUgCuGuCgGaGcCTsT 1201 GGCfUfCfCfGGCfAGCfAGAUfGGCfTsT 1202 AD- 3015115 3480-3498 GfcCfaUfcUfgCfuGfcCfgGfaGfcCfTsT 1203GGCUCauGCagCAGAUggcTsT 1204 AD- 54 15125 3480-3498GCfCfAUfCfUfGCfUfGCfCfGGAGCfCfTsT 1205 GGCGCauGCagCAGAUggcTsT 1206 AD-51 15135 3480-3498 GcCaUcUgCuGcCgGaGcCTsT 1207 GGCUCauGCagCAGAUggcTsT1208 AD- 49 15145 3481-3499 CAUUUAUCUUUUGGGUCUGTsT 1209CAGACCCAAAAGAUAAAUGTsT 1210 AD- 49 61 9578 3481-3499cAuuuAucuuuuGGGuuuGTsT 1211 cAGACCcAAAAGAuAAAUGTsT 1212 AD- 111 97043485-3403 UAUCUUUUGGGUCUGUCCUTsT 1213 AGGACAGACCCAAAAGAUATsT 1214 AD- 669558 3485-3503 uAucuuuuGGGucuGuccuTsT 1215 AGGAcAGACCcAAAAGAuATsT 1216AD- 63 9684 3504-3522 CUCUGUUGCCUUUUUACAGTsT 1217 CUGUAAAAAGGCAACAGAGTsT1218 AD- 29 30 9634 3504-3522 cucuGuuGccuuuuuuAcAGTsT 1219CUGuAAAAAGGcAAcAGAGTsT 1220 AD- 14 27 9760 3512-3530CCUUUUUACAGCCAACUUUTT 1221 AAAGUUGGCUGUAAAAAGGTT 1222 AD- 5 154113521-3539 AGCCAACUUUUCUAGACCUTT 1223 AGGUCCAGAAAAGUUGGCUTT 1224 AD- 2315266 3526-3544 ACUUUUCUAGACCUGUUUUTT 1225 AAAACAGGUCUAGAAAAGUTT 1226AD- 12 15382 3530-3548 UUCUAGACCUGUUUUGCUUTsT 1227AAGCAAAACAGGUCUAGAATsT 1228 AD- 23 24 9554 3530-3548uucuAGAccuGuuuuGcuuTsT 1229 AAGcAAAACcGGUCuAGAATsT 1230 AD- 12 22 0.100.10 9680 3530-3548 UfuCfuAfgAfcCfuGfuUfuGfgCfuGfTsT 1231p-aAfgCfaafaafcAfgGfuCfuAfgAfaTsT 1232 AD- 12 14676 3530-3548UfCfCfUfAGACfCfUfGUfUfUfUfGCfUfUTTs 1233 AAGTAAAACfAGGUfCfUfAGAATsT 1234AD- 13 T 14686 3530-3548 UuCuAgAcCuGcUuUgCuUTsT 1235p-aAfgCfaAfaAfcAfgGfaCfcAfgAfaTsT 1236 AD- 12 14696 3530-3548UuCuAgAcCuGuUuUgCuUTsT 1237 AAGCfAAAACfAGGUfCfUfAGAATsT 1238 AD- 1814706 3530-3548 UfuCfuAfgAfcCfuGfuUfaUfTCfuUTTsT 1239AAGcAaaACagGUCUAgaaTsT 1240 AD- 17 14716 3530-3548UfUfCfUfAGACfCfUfGUfUfUfUfGCfUfUTTs 1241 AAGcAaaACagGUCUAgaaTsT 1242 AD-16 T 14726 3530-3548 UuCuAgAcCuGcUuUgCuUTsT 1243 AAGcAaaACagGUCUAgaaTsT1244 AD- 9 14736 3530-3548 CfaUfaGfgCfcUfgGfaGfcUfaAfuCTTsT 1245p-aAfuAfaAfcUfcCfaFfgCfcUfaUfgTsT 1246 AD- 27 15082 3530-3548CfAUfAGGCfCfUfGGAGUfUfUfACfUTTsT 1247 AAUfAAACfUfCfCfAGGCfCfUTAUfGTsT1248 AD- 28 15092 3530-3548 CaUaGgCcUgGaGuUuAuUTsT 1249p-aAfcAfaAfcUfcCfaGfgCfcUfaCfgTsT 1250 AD- 19 15102 3530-3548CaUaGgCcUgGaGuUcAuUTsT 1251 AAUfAAACfUfCTCfAGGCfCTUfAUTGTsT 1252 AD- 1715112 3530-3548 CfaUfaGfgCfcUfgGfaGfcUfuAfuUfTsT 1253AAUAAacCCcaGGCCUaugTsT 1254 AD- 56 15122 3530-3548CfAUfAGGCfCfUfGGAGUfUfUfACfUTTsT 1255 AAUAAucUCcaGGCCUaugTsT 1256 AD- 3915132 3530-3548 CaUaGgCcUgGaGuUuAuUTsT 1257 AAUAAacUCcaGGCCCaugTsT 1258AD- 46 15142 3531-3549 UCUAGACCUGUUUUGCUUUTsT 1259AAAGCAAAACAGGUCUAGATsT 1260 AD- 27 22 0.02 9553 3531-3549ucuAGAccuGuuuuGccuuTsT 1261 AAAGcAAAAcAGGUCuAGATsT 1262 AD- 17 21 96793531-3549 UfcUfaGfaCfcUfgUfuUfuGfcUfuUfTsT 1263p-aAfaGfcAfaAfaCfaGfgUfcUfaGfaTsT 1264 AD- 11 14675 3531-3549UfCfUfAGACfCfUfGUfUfGfUfGCfUfUfUfTs 1265 AAAGCfAAAACfAGGUfCfUfAGATsT1266 AD- 19 T 14685 3531-3549 UcUaGaCcUgUuUuGcUuUTsT 1267p-aAfaGfcAfaAfaCfaGfgUfcUfaGfaTsT 1268 AD- 12 14695 3531-3549UcUaGaCcUgUuUuGcUuUTsT 1269 AAAGCfAAAACfAGGUfCfUfAGATsT 1270 AD- 1614705 3531-3549 UfcUfaGfaCfcUfgUfuUfuGfcUfaUfTsT 1271AAAGCaaAAcaGGUCUaGATsT 1272 AD- 19 14715 3531-3549UfCfUfAGACfCfUfGUfUfUfUfGCfUfUfUfTs 1273 AAAGCaaAAcaGGUCUagaTsT 1274 AD-19 T 14725 3531-3549 UcUaGaCcUgUuUuGcUuUTsT 1275 AAAGCaaAAcuGGUCUagaTsT1276 AD- 19 14735 3531-3549 UfcAfuAfgGfccfuGfgAfgUfuUfaUTTsT 1277p-aUfaAfaCfuCfcAfgGfcCfuAfuGfaTsT 1278 AD- 30 15081 3531-3549UfCfAUfAGGCfCfUfGGAGUfUfUfAUfTsT 1279 AUfAAACfUfCfCfAGGCfCfUfAUfGATsT1280 AD- 16 15091 3531-3549 UcAuAgGcCuGgAgUuUaUTsT 1281p-aUfAAfaCfuCfcAfgGfcCfuAgcGfuTsT 1282 AD- 16 15101 3531-3549UcAuAgGcCuGgAgUuUaUTsT 1283 AUfAAAcFufCfCtAGGCfCfUfAUfGATsT 1284 AD- 1115111 3531-3549 UfcAfuAfgGfcCfuGfgAfgUfaUfAUfTsT 1285AUAAAcuCCagGCCUAugaTsT 1286 AD- 19 15121 3531-3549UfCfAUfAGGCfCfUfGGAGUfUfUTAUfTsT 1287 AUAAAcuCCagGCCUAugaTsT 1288 AD- 1715131 3531-3549 UcAuAgGcCuGgAgUuUaUTsT 1289 AUAAAcuCCagGCCUAugaTsT 1290AD- 18 15141 3557-3575 UGAAGAUAUUUAGUCCGGGTsT 1291CCCAGAAUAAAUAUCUUCATsT 1292 AD- 97 68 9626 3557-3575uGAAGAuAuuuAuuuuGGGTsT 1293 CCcAGAAuAAAuAUCUUcATsT 1294 AD- 28 33 97523570-3588 UCUGGGUUUUGUAGCAGUUTsT 1295 AAAUGCUACAAAACCCAGATsT 1296 AD- 2324 9629 3570-3588 ucuGGGuuuuGuAGcAuuuTsT 1297 AAAUGCuAcAAAACCcAGATsT1298 AD- 28 29 9755 3613-3631 AUAAAAACAAACAAACGUUTT 1299AACGUUUGUUUGUUUUUAUTT 1300 AD- 21 15412 3677-3635 AAACAAACAAACGUUGUCCTT1301 GGACAACGUUUGUUUGUUUTT 1302 AD- 73 15211 3618-3636AACAAACAAACGUUGUCCUTT 1303 AGGACAACGUUGUUUGUUTT 1304 AD- 41 15300 ¹U, C,A, G: corresponding ribonucleotide; T: deoxythynidine; u, c, a, g:corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding2′-deoxy-2′fluoro ribonucleotide; where nucleotides are written insequence, they are connected by 3′-5′ phosophodiester groups;nucleotides with interjected “s” are connected by 3′-O-5′-Ophosphorothiodiester groups; unless denoted by prefix“p”,oligonucleotides are devoid of a 5′-phosphate group on the 5′-mostnucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide

TABLE 2 Remaining mRNA in % of controls SEQ SEQ at siRNA Duplex IDAntisense-strand ID conc. of 30 number Sense strand sequence (5′-3′) NO:sequence (5′-3′) NO: nM AD-10792 GccuGGAGuuuAuucGGATTsT 1305UUCCAAuAAACUCcAGGCTsT 1306 15 AD-10793 GccuGGAGuuuAuucGGAATsT 1307uUcCGAAuAAACUccAGGCTsT 1308 32 AD-10796 GccuGGAGuuuAuucGGAATsT 1309UUCCGAAUAAACUCCAGGCTsT 1310 13 AD-12038 GccuGGAGuuuAuucGGAATsT 1311uUCCGAAUAAACUCCAGGCTsT 1312 13 AD-12039 GccuGGAGuuuAuucGGAATsT 1313UuCCGAAUAAACUCCAGGCTsT 1314 29 AD-12040 GccuGGAGuuuAuucGGAATsT 1315UUcCGAAUAAACUCCAGGCTsT 1316 10 AD-12041 GccuGGAGuuuAuucGGAATsT 1317UUCcGAAUAAACUCCAGGCTsT 1318 11 AD-12042 GCCUGGAGUUUAUUCGGAATsT 1319uUCCGAAUAAACUCCAGGCTsT 1320 12 AD-12043 GCCUGGAGUUUAUUCGGAATsT 1321UuCCGAAUAAACUCCAGGCTsT 1322 13 AD-12044 GCCUGGAGUUUAUUCGGAATsT 1323UUcCGAAUAAACUCCAGGCTsT 1324 7 AD-12045 GCCUGGAGUUUAUUCGGAATsT 1325UUCcGAAUAAACUCCAGGCTsT 1326 8 AD-12046 GccuGGAGuuuAuucGGAA 1327UUCCGAAUAAACUCCAGGCscsu 1328 13 AD-12047 GccuGGAGuuuAuucGGAAA 1329UUUCCGAAUAAACUCCAGGCscsu 1330 17 AD-12048 GccuGGAGuuuAuucGGAAAA 1331UUUUCCGAAUAAACUCCAGGCscsu 1332 43 AD-12049 GccuGGAGuuuAuucGGAAAAG 1333CUUUUCCGAAUAAACUCCAGGCscsu 1334 34 AD-12050 GccuGGAGuuuAuucGGAATTab 1335UUCCGAAUAAACUCCAGGCTTab 1336 16 AD-12051 GccuGGAGuuuAuucGGAAATTab 1337UUUCCGAAuAAACUCCAGGCTTab 1338 31 AD-12052 GccuGGAGuuuAuucGGAAAATTab 1339UUUUCCGAAUAAACUCCAGGCTTab 1340 81 AD-12053 GccuGGAGuuuAuucGGAAAAGTTab1341 CUUUUCCGAAUAAACUCCAGGCTTab 1342 46 AD-12054 GCCUGGAGUUUAUUCGGAATsT1343 UUCCGAAUAAACUCCAGGCscsu 1344 8 AD-12055 GccuGGAGuuuAuucGGAATsT 1345UUCCGAAUAAACUCCAGGCscsu 1346 13 AD-12056 GcCuGgAgUuUaUuCgGaA 1347UUCCGAAUAAACUCCAGGCTTab 1348 11 AD-12057 GcCuGgAgUuUaUuCgGaA 1349UUCCGAAUAAACUCCAGGCTsT 1350 8 AD-12058 GcCuGgAgUuUaUuCgGaA 1351UUCCGAAuAAACUCcAGGCTsT 1352 9 AD-12059 GcCuGgAgUuUaUuCgGaA 1353uUcCGAAuAAACUccAGGCTsT 1354 23 AD-12060 GcCuGgAgUuUaUuCgGaA 1355UUCCGaaUAaaCUCCAggc 1356 10 AD-12061 GcCuGgnAgUuUaUuCgGaATsT 1357UUCCGaaUAaaCUCCAggcTsT 1358 7 AD-12062 GcCuGgAgUuUaUuCgGaATTab 1359UUCCGaaUAaaCUCCAggcTTab 1360 10 AD-12063 GcCuGgAgUuUaUuCgGaA 1361UUCCGaaUAaaCUCCAggcscsa 1362 19 AD-12064 GcCuGgnAgUuUaUuCgGaATsT 1363UUCCGAAuAAACUCcAGGCTsT 1364 15 AD-12065 GcCuGgAgUuUaUuCgGaATTab 1365UUCCGAAuAAACUCcAGGCTTab 1366 16 AD-12066 GcCuGgAgUuUaUuCgGaA 1367UUCCGAAuAAACUCcAGGCscsu 1368 20 AD-12067 GcCuGgnAgUuUaUuCgGaATsT 1369UUCCGAAUAAACUCCAGGCTsT 1370 17 AD-12068 GcCuGgAgUuUaUuCgGaATTab 1371UUCCGAAUAAACUCCAGGCTTab 1372 18 AD-12069 GcCuGgAgUuUaUuCgGaA 1373UUCCGAAUAAACUCCAGGCscsu 1374 13 AD-12338 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf1375 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1376 15 AD-12339 GcCuGgAgUuUaUuCgGaA1377 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1378 14 AD-12340 GccuGGAGuuuAuucGGAA1379 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1380 19 AD-12341GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1381 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc1382 12 TsT AD-12342 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1383UUCCGAAuAAACUCcAGGCTsT 1384 13 AD-12343 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT1385 uUcCGAAuAAACUccAGGCTsT 1386 24 AD-12344GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1387 UUCCGAAUAAACUCCAGGCTsT 1388 9AD-12345 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1389 UUCCGAAUAAACUCCAGGCscsu1390 12 AD-12346 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1391UUCCGaaUAaaCUCCAggcscsu 1392 13 AD-12347 GCCUGGAGUUUAUUCGGAATsT 1393P-uUfcCfgAgaUfaAfaCfuCfcAfgGfc 1394 11 TsT AD-12348GccuGGAGuuuAuucGGAATsT 1395 P-uUfcCfgAgaUfaAfaCfuCfcAfgGfc 1396 8 TsTAD-12349 GcCuGgnAgUuUaUuCgGaATsT 1397 P-uUfcCfgAgaUfaAfaCfuCfcAfgGfc1398 11 TsT AD-12350 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTTab 1399P-uUfcCfgAgaUfaAfaCfuCfcAfgGfc 1400 17 TTab AD-12351GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1401 P-uUfcCfgAgaUfaAfaCfuCfcAfgGfcs 140211 Cfsu AD-12352 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1403UUCCGaaUAaaCUCCAggcscsu 1404 11 AD-12354 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf1405 UUCCGAAUAAACUCCAGGCscsu 1406 11 AD-12355GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1407 UUCCGAAuAAACUCcAGGCTsT 1408 9AD-12356 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1409 uUcCGAAuAAACUccAGGCTsT 141025 AD-12357 GmocCmouGmogAm02gUmouUmoaUmouCmogGmoaA 1411UUCCGaaUAaaCUCCAggc 1412 56 AD-12358GmocCmouGmogAm02gUmouUmoaUmouCmogGmoaA 1413P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1414 29 AD-12359GmocCmouGmogAm02gUmouUmoaUmouCmogGmoaA 1415P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcs 1416 30 Cfsu AD-12360GmocCmouGmogAm02gUmouUmoaUmouCmogGmoaA 1417 UUCCGAAUAAACUCCAGGCscsu 141815 AD-12361 GmocCmouGmogAm02gUmouUmoaUmouCmogGmoaA 1419UUCCGAAuAAACUCcAGGCTsT 1420 20 AD-12362GmocCmouGmogAm02gUmouUmoaUmouCmogGmoaA 1421 uUcCGAAuAAACUccAGGCTsT 142251 AD-12365 GmocCmouGmogAm02gUmouUmoaUmouCmogGmoaA 1423UUCCGaaUAaaCUCCAggcscsu 1424 11 AD-12364GmocCmouGmogAmogUmouUmoaUmonCmogGmonATsT 1425 UUCCGaaUAaaCUCCAggcTsT1426 25 AD-12365 GmocCmouGmogAmogUmouUmoaUmouCmogGmonATsT 1427UUCCGAAuAAACUCcAGGCTsT 1428 18 AD-12366GmocCmouGmogAmogUmouUmoaUmouCmogGmonATsT 1429 UUCCGAAUAAACUCCAGGCTsT1430 23 AD-12367 GmocmocmouGGAGmoumoumouAmoumoumocGGAATsT 1431UUCCGaaUAaaCUCCAggcTsT 1432 42 AD-12368GmocmocmouGGAGmoumoumonAmoumoumocGGAATsT 1433 UUCCGAAuAAACUCcAGGCTsT1434 40 AD-12369 GmocmocmouGGAGmoumoumouAmoumoumocGGAATsT 1435UUCCGAAUAAACUCCAGGCTsT 1436 26 AD-12370GmocmocmouGGAGmoumoumouAmoumoumocGGAATsT 1437P-UfUfCfCfGAAUfAAACfUfCfCfAGGCf 1438 68 TsT AD-12371GmocmocmouGGAGmoumoumouAmoumoumocGGAATsT 1439P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfs 1440 60 CfsUf AD-12372GmocmocmouGGAGmoumoumonAmoumoumocGGAATsT 1441P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcs 1442 60 Cfsu AD-12373GmocmocmouGGAGmoumoumouAmoumoumocGGAATsT 1443 UUCCGAAUAAACUCCAGGCTsT1444 55 AD-12374 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1445UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1446 9 AD-12375GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1447 UUCCGAAUAAACUCCAGGCTsT 1448 16AD-12377 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1449 uUcCGAAuAAACUccAGGCTsT1450 88 AD-12378 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1451UUCCGaaUAaaCUCCAggcscsu 1452 6 AD-12379 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT1453 UUCCGAAUAAACUCCAGGCscsu 1454 6 AD-12380GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1455 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcs1456 8 Cfsu AD-12381 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1457P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1458 10 TsT AD-12382GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1459 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCf1460 7 TsT AD-12383 GCCUGGAGUUUAUUCGGAATsT 1461P-UfUfCfCfGAAUfAAACfUfCfCfAGGCf 1462 7 TsT AD-12384GccuGGAGuuuAuucGGAATsT 1463 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCf 1464 8 TsTAD-12385 GcCuGgnAgUuUaUuCgGaATsT 1465 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCf146 8 TsT AD-12386 GfcCfuGfgAfgUfuUfaUfuCfuGfaAf 1467P-UfUfCfCfGAAUfAAACfUfCfCfAGGCf 1468 11 TsT AD-12387GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1469 UfUfCfCfGAAUfAAACfUfCfCfAGGCfs 147013 CfsUf AD-12388 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1471P-uUfcCfgAfaUfaAfaCfuCgcAfgGfc 1472 19 AD-12389GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1473 P-uUfcCfgAfaUfaAfaCfuCgcAfgGfcs 147416 Cfsu AD-12390 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1475UUCCGAAUAAACUCCAGGCscsu 1476 17 AD-12391 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA1477 UUCCGaaUAaaCUCCAggc 1478 21 AD-12392 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA1479 UUCCGAAUAAACUCCAGGCTsT 1480 28 AD-12393GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1481 UUCCGAAuAAACUCcAGGCTsT 1482 17AD-12394 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1483 uUcCGAAuAAACUccAGGCTsT 148475 AD-12395 GmocCmouGmogAmogUmouUmoaUmouCmogGmoaATsT 1485P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfs 1486 55 CfsUf AD-12396GmocCmouGmogAm02gUmouUmoaUmouCmogGmoaA 1487P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfs 1488 59 CfsUf AD-12397GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1489 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfs 149020 CfsUf AD-12398 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1491P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfs 1492 11 CfsUf AD-12399GcCuGgnAgUuUaUuCgGaATsT 1493 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfs 1494 13CfsUf AD-12400 GCCUGGAGUUUAUUCGGAATsT 1495P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfs 1496 12 CfsUf AD-12401GccuGGAGuuuAuucGGAATsT 1497 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfs 1498 13CfsUf AD-12402 GccuGGAGuuuAuucGGAA 1499 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfs1500 14 CfsUf AD-12403 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1501P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfs 1502 4 CfsUf AD-9314GCCUGGAGUUUAUUCGGAATsT 1503 UUCCGAAUAAACUCCAGGCTsT 1504 9 ¹U, C, A, G:corresponding ribonucleotide; T: deoxythymidine; u, c, a, g:corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding2′-deoxy-2′-fluoro ribonucleotide; moc, mou, mog, moa: corresponding2′-MOE nucleotide; where nucleotides are written in sequence, they areconnected by 3′-5′ phosphodiester groups; ab: 3′-terminal abasicnucleotide; nucleotides with interjected “s” are connected by 3′-O-5′-Ophosphorothiodiestergroups; unless denoted by prefix “p-”,oligonucleotides are devoid of a 5′-phosphate group on the 5′-mostnucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting theexpression of a human proprotein convertase subtilisin kexin 9 (PCSK9)gene in a cell, wherein said dsRNA comprises at least two sequences thatare complementary to each other and wherein a sense strand comprises afirst sequence and an antisense strand comprises a second sequencecomprising at least 15 contiguous nucleotides of SEQ ID NO:1230.
 2. ThedsRNA of claim 1, wherein said first sequence comprises SEQ ID NO:1229and said second sequence comprises SEQ ID NO:1230.
 3. The dsRNA of claim1, wherein said dsRNA comprises at least one modified nucleotide.
 4. ThedsRNA of claim 2, wherein said dsRNA comprises at least one modifiednucleotide.
 5. The dsRNA of claim 3, wherein said modified nucleotide ischosen from the group of: a 2′-O-methyl modified nucleotide, anucleotide comprising a 5′-phosphorothioate group, and a terminalnucleotide linked to a cholesteryl derivative or dodecanoic acidbisdecylamide group.
 6. The dsRNA of claim 3, wherein said modifiednucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anabasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modifiednucleotide, morpholino nucleotide, a phosphoramidate, and a non-naturalbase comprising nucleotide.
 7. The dsRNA of claim 1, wherein said sensestrand consists of SEQ ID NO:1229 and said antisense strand consists ofSEQ ID NO:1230.
 8. The dsRNA of claim 6, wherein said first sequencecomprises SEQ ID NO:1229 and said second sequence comprises SEQ IDNO:1230.
 9. A cell comprising the dsRNA of claim
 1. 10. A pharmaceuticalcomposition for inhibiting the expression of the proprotein convertasesubtilisin kexin 9 (PCSK9) gene in an organism, comprising a dsRNA and apharmaceutically acceptable carrier, wherein the dsRNA comprises atleast two sequences that are complementary to each other and wherein asense strand comprises a first sequence and an antisense strandcomprises a second sequence comprising at least 15 contiguousnucleotides of SEQ ID NO:1230 and wherein said dsRNA is capable ofcausing a decrease in serum lipid levels.
 11. The pharmaceuticalcomposition of claim 10, wherein said first sequence of said dsRNAcomprises SEQ ID NO:1229, and said second sequence of said dsRNAcomprises SEQ ID NO:1230.
 12. A vector for inhibiting the expression ofa proprotein convertase subtilisin kexin 9 (PCSK9) gene in a cell, saidvector comprising a regulatory sequence operably linked to a nucleotidesequence that encodes at least one strand of the dsRNA of claim
 1. 13. Acell comprising the vector of claim
 12. 14. The dsRNA of claim 1,wherein said dsRNA, upon contact with a cell expressing said PCSK9 gene,inhibits expression of said PCSK9 gene.
 15. The dsRNA of claim 14,wherein said contact is performed in vitro at 30 nM or less.
 16. ThedsRNA of claim 1, wherein said dsRNA, upon contact with HepG2 cellsexpressing PCSK9, inhibits expression of said PCSK9 gene by at least20%.
 17. The pharmaceutical composition of claim 10, wherein said dsRNA,upon contact with a cell expressing said PCSK9, inhibits expression ofsaid PCSK9 gene.
 18. The dsRNA of claim 1, wherein administration ofsaid dsRNA to an animal results in a decrease in total serumcholesterol.
 19. The pharmaceutical composition of claim 10, whereinadministration of said pharmaceutical composition to an animal resultsin a decrease in total serum cholesterol.
 20. The dsRNA ofpharmaceutical composition of claim 10, wherein said sense strandconsist of SEQ ID NO:1229 and said antisense strand consist of SEQ IDNO:1230.